Regulation of angiogenesis with zinc finger proteins

ABSTRACT

Provided herein are a variety of methods and compositions for regulating angiogenesis, such methods and compositions being useful in a variety of applications where modulation of vascular formation is useful, including, but not limited to, treatments for ischemia and wound healing. Certain of the methods and compositions accomplish this by using various zinc finger proteins that bind to particular target sites in one or more VEGF genes. Nucleic acids encoding the zinc finger proteins are also disclosed. Methods for modulating the expression of one or more VEGF genes with the zinc finger proteins and nucleic acids are also disclosed. Such methods can also be utilized in a variety of therapeutic applications that involve the regulation of endothelial cell growth. Pharmaceutical compositions including the zinc finger proteins or nucleic acids encoding them are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of copending U.S. applicationSer. No. 09/846,033, filed Apr. 30, 2001, which is acontinuation-in-part of U.S. application Ser. No. 09/736,083, filed Dec.12, 2000, which is a continuation-in-part of U.S. application Ser. No.09/733,604, filed Dec. 7, 2000, all of which are incorporated herein byreference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

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REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

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BACKGROUND

The development of the vascular system (sometimes referred to as thevascular tree) involves two major processes: vasculogenesis andangiogenesis. Vasculogenesis is the process by which the major embryonicblood vessels originally develop from early differentiating endothelialcells such as angioblasts and hematopoietic precursor cells that in turnarise from the mesoderm. Angiogenesis is the term used to refer to theformation of the rest of the vascular system that results from vascularsprouting from the pre-existing vessels formed during vasculogenesis(see, e.g., Risau et al. (1988) Devel. Biol., 125:441-450). Bothprocesses are important in a variety of cellular growth processesincluding developmental growth, tissue regeneration and tumor growth, asall these processes require blood flow for Given its key role in bothnormal physiological and pathological processes, not surprisinglyconsiderable research effort has been directed towards identifyingfactors involved in the stimulation and regulation of angiogenesis. Anumber of growth factors have been purified and characterized. Suchfactors include fibroblast growth factors (FGFs), platelet-derivedgrowth factor (PDGF), transforming growth factor alpha (TGFα), andhepatocyte growth factor (HGF) (for reviews of angiogenesis regulators,see, e.g., Klagsbrun et al. (1991) Ann. Rev. Physiol., 53:217-39; andFolkman et al. (1992) J. Biol. Chem., 267:10931-934) the delivery ofnecessary nutrients.

Thus, angiogenesis plays a critical role in a wide variety offundamental physiological processes in the normal individual includingembryogenesis, somatic growth, and differentiation of the nervoussystem. In the female reproductive system, angiogenesis occurs in thefollicle during its development, in the corpus luteum followingovulation and in the placenta to establish and maintain pregnancy.Angiogenesis additionally occurs as part of the body's repair processes,such as in the healing of wounds and fractures. Thus, promotion ofangiogenesis can be useful in situations in which establishment orextension of vascularization is desirable. Angiogenesis, however, isalso a critical factor in a number of pathological processes, perhapsmust notably tumor growth and metastasis, as tumors require continuousstimulation of new capillary blood vessels in order to grow. Otherpathological processes affected by angiogenesis include conditionsassociated with blood vessel proliferation, especially in thecapillaries, such as diabetic retinopathy, arthropathies, psoriasis andrheumatoid arthritis.

Current research indicates that a family of endothelial cell-specificgrowth factors, the vascular endothelial growth factors (VEGFs),together with their cognate receptors, are primarily responsible forstimulation of endothelial cell growth and differentiation. Thesefactors are members of the PDGF family and appear to act primarily viaendothelial receptor tyrosine kinases (RTKs).

The first identified and most well studied member of this particularfamily is the vascular endothelial growth factor (VEGF), also referredto as VEGF-A. This particular growth factor is a dimeric glycoprotein inwhich the two 23 kD subunits are joined via a disulfide bond. FiveVEGF-A isoforms encoded by distinct mRNA splice variants appear to beequally effective in stimulating mitogenesis in endothelial cells, buttend to have differing affinities for cell surface proteoglycans.

VEGF-A acts to regulate the generation of new blood vessels duringembryonic vasculogenesis and then subsequently plays an important rolein regulating angiogenesis later in life. Studies showing thatinactivation of a single VEGF-A allele results in embryonic lethalityprovide evidence as to the significant role this protein has in vasculardevelopment and angiogenesis (see, e.g., Carmeliet et al. (1996) Nature380: 435-439; and Ferrara et al. (1996) Nature, 380: 439-442). VEGF-Ahas also been shown to have other activities including a strongchemoattractant activity towards monocytes, the ability to induce theplasminogen activator and the plasminogen activator inhibitor inendothelial cells, and to induce microvascular permeability. VEGF-A issometimes also referred to as vascular permeability factor (VPF) in viewof this latter activity. The isolation and properties of VEGF-A havebeen reviewed (see, e.g., Ferrara et al. (1991) J. Cellular Biochem. 47:211-218; and Connolly, J. (1991) J. Cellular Biochem. 47:219-223).

Alternative mRNA splicing of a single VEGF-A gene gives rise to at leastfive isoforms of VEGF-A. These isoforms are referred to as VEGF-A121;VEGF-A145; VEGF-A165; VEGF-A189; and VEGF-A206. As the name implies, theVEGF-A165 isoform is a 165 amino acid species and has a molecular weightof approximately 46 kD; this isoform is the predominant molecular formfound in normal cells and tissues. VEGF-A165 includes a 44 amino acidregion near the carboxyl-terminal region that is enriched in basic aminoacid residues. It also exhibits an affinity for heparin and heparinsulfates.

VEGF-A121 is the shortest form, with a deletion of 44 amino acidsbetween positions 116 and 159 as compared to the VEGF A165 isoform. Itis freely diffusible in the surrounding extracellular matrix. VEGF-A 189is a longer form with an insertion of 24 highly basic residues atposition 116 with respect to VEGF A165. The VEGF-A206 isoform includesinsertion of 41 amino acids with respect to the VEGF A165 isoform,including the 24 amino acid insertion found in VEGF-A189. VEGF-A121 andVEGF-A165 are soluble proteins, with VEGF-A165 being the predominantisoform secreted by cells. In contrast, VEGF-A189 and VEGF-A206 appearto be mostly cell-associated. All of these isoforms of VEGF-A arebiologically active.

VEGF-B, also referred to as VRF, has similar angiogenic and otherproperties to those of VEGF-A, but is distributed and expressed intissues differently from VEGF-A. In particular, VEGF-B is very stronglyexpressed in heart, and only weakly in lung, whereas the reverse is thecase for VEGF-A. This suggests that VEGF-A and VEGF-B, despite the factthat they are co-expressed in many tissues, may have functionaldifferences. The amino acid sequence of VEGF-B is approximately 44%identical to that of VEGF-A. Alternative exon splicing of the VEGF-Bgene generates two isoforms encoding human proteins of 167 and 186 aminoacids, and referred to as VEGF-B167 and VEGF-B186, respectively.VEGF-B167 tends to remain cell-associated, while VEGF-B186 is freelysecreted. The isolation and characteristics of these isoforms arediscussed in PCT/US96/02957 and in Olofsson et al. (1996) Proc. Natl.Acad. Sci. USA 93: 2576-2581.

VEGF-C is also referred to as VEGF-related protein (hence VRP) orVEGF-2. The protein is roughly 30% identical to the amino acid sequenceof VEGF-A, and includes N-terminal and C-terminal extensions not presentin VEGF-A, VEGF-B or P1GF (infra). Although the protein induces vascularpermeability and promotes endothelial growth, it is less potent thanVEGF-A. Its isolation and characteristics are disclosed in Joukov et al.(1996) EMBO J. 15: 290-298.

VEGF-D was isolated from a human breast cDNA library, commerciallyavailable from Clontech, by screening with an expressed sequence tagobtained from a human cDNA library designated “Soares Breast 3NbHBst” asa hybridization probe (Achen et al. (1998) Proc. Natl. Acad. Sci. USA95:549-553). The protein is also referred to as FIGF, for c-fos-inducedgrowth factor. The VEGF-D gene is expressed most abundantly in lung,heart, small intestine and fetal lung. It is found at lower levels inskeletal muscle, colon and pancreas. Its isolation and characteristicsare discussed in PCT Publication WO 98/07832.

Recently several additional VEGF-like proteins have been identified fromvarious strains of orf viruses; in the literature these viral VEGFproteins have sometimes been collectively referred to as VEGF-E. Oneprotein, variously referred to as OV NZ2, ORFV2-VEGF, OV-VEGF2, andVEGF-ENZ2 has been isolated from the orf viral strain NZ2 (see, e.g.,Lyttle, D. J. et al. (1994) J. Virology 68:84-92; and PCT Publication WO00/25805). Another viral VEGF-like protein referred to as NZ7, OV-VEGF7,VEGF-E and VEGF-ENZ7 has been found in the NZ7 strain of orf viruses.This protein exhibits potent mitogenic activity but lacks the basicdomain of certain VEGF proteins such as VEGF-A165 (see, e.g., Lyttle, D.J. et al. (1994) J. Virology 68:84-92; and Ogawa, S. et al. (1998) J.Biol. Chem.

273:31273-31282). A third VEGF-like protein has been identified in a NZstrain, specifically a NZ10 strain and is referred to simply as NZ10(see, e.g., PCT Publication WO 00/25805). Yet another VEGF-like proteinhas been identified in the orf virus strain D1701 and in some instanceshas been referred to as VEGF-ED1701 (see, e.g., Meyer, M. et al. (1999)EMBO J. 18:363-74).

In addition to these viral VEGF-E genes, a VEGF-like growth factorisolated from mammalian sources has also been named VEGF-E. Theisolation and characterization of this VEGF-like factor is discussed inPCT Publication WO 99/47677.

Another VEGF-like protein has been termed PDGF/VEGF-Like Growth FactorH, or simply VEGF-H. It is discussed in PCT Publication WO 00/44903.Additional VEGF-like proteins include one called VEGF-R (see, e.g., PCTPublication WO 99/37671) and another referred to as VEGF-X (see, e.g.,PCT publication WO 00/37641). Most recently a VEGF protein referred toas VEGF-138 has been identified by Neufeld and others. A final proteinrelated to the VEGF proteins is the Placenta Growth Factor, P1GF. Thisprotein was isolated from a term placenta cDNA library. A segment of theprotein exhibits high levels of homology with VEGF-A. Its isolation andcharacteristics are described by Maglione et al. (1991) Proc. Natl.Acad. Sci. USA 88:9267-9271. Its biological function is presently notwell understood. Two alternatively transcribed mRNAs have beenidentified in humans (P1GF-1 and P1GF-2).

The foregoing PDGF/VEGF family members act primarily by binding toreceptor tyrosine kinases. Five endothelial cell-specific receptortyrosine kinases have been identified thus far, namely VEGFR-1 (alsocalled Flt-1), VEGFR-2 (also called KDR/Flk-1), VEGFR-3 (Flt4), Tie andTek/Tie-2. Each of these kinases have the tyrosine kinase activitynecessary for signal transduction. The essential, specific role invasculogenesis and angiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie andTek/Tie-2 has been demonstrated by targeted mutations inactivating thesereceptors in mouse embryos.

VEGF-A binds VEGFR-1 and VEGFR-2 with high affinity, as well asneurophilin 1. As just indicated, VEGFR-1 binds VEGF-A, but also bindsVEGF-B and P1GF. VEGF-C has been shown to be the ligand for VEGFR-3, andit also activates VEGFR-2 (Joukov et al. (1996) The EMBO Journal 15:290-298). Both VEGFR-2 and VEGFR-3 bind VEGF-D. Initial studies with theviral VEGF proteins (i.e., the viral VEGF-E group) show that theseproteins selectively bind VEGFR-2 but not VEGFR-1 (see, e.g., Ogawa, S.et al. (1998) J. Biol. Chem. 273:31273-31282; and Meyer, M. et al.(1999) 18:363-74). A ligand for Tek/Tie-2 has been described in PCTPublication WO 96/11269. The ligand for Tie has not yet been identified.Additional details regarding the various VEGF receptors are provided inPCT Publication WO 00/25805.

Recently, a 130-135 kDa VEGF isoform specific receptor has been purifiedand cloned (Soker et al. (1998) Cell 92:735-745). The evidence indicatesthat this VEGF receptor specifically binds to the VEGF165 isoform viathe exon 7 encoded sequence of VEGF165, which sequence shows weakaffinity for heparin (Soker et al. (1998) Cell, 92:735-745). Thereceptor has also been found to be identical to human neurophilin-1(NP-1), a receptor involved in early stage neuromorphogenesis. One ofthe splice variants of P1GF, namely P1GF-2, also appears to interactwith NP-1 (Migdal et al., (1998) J. Biol. Chem. 273: 22272-22278).

Thus, a variety of cell growth factors, in particular VEGF proteins andVEGF-related proteins, have been identified. Certain receptors that bindto the VEGF proteins have also been identified. However, modulation ofthe expression of VEGF proteins and VEGF-related proteins so as tomodulate the process of angiogenesis has not been described. The abilityto modulate the process of angiogenesis in a cell or group of cells,using one or more exogenous molecules, would have utility in activatingbeneficial aspects associated with endothelial cell growth and inrepressing non-beneficial aspects.

SUMMARY

A variety of zinc finger proteins (ZFPs) and methods utilizing suchproteins are provided for use in regulating gene expression. Certain ofthe ZFPs are designed to bind to specific target sequences within genesand thereby modulate angiogenesis. The ZFPs can be fused to a regulatorydomain as part of a fusion protein. By selecting either an activationdomain or repressor domain for fusion with the ZFP, one can eitheractivate or repress gene expression. Thus, by appropriate choice of theregulatory domain fused to the ZFP, one can selectively modulate theexpression of a gene and hence various physiological processescorrelated with such genes. Thus, with angiogenesis, for example, byattaching an activation domain to a ZFP that binds to a target sequencewithin a gene that affects angiogenesis, one can enhance certainbeneficial aspects associated with angiogenesis (e.g., alleviation ofischemia). In contrast, if angiogenesis is associated with harmfulprocesses (e.g., delivery of blood supply to tumors) one can reduceangiogenesis by using ZFPs that are fused to a repressor. Hence, bindingof this type of ZFP to a gene involved in angiogenesis can significantlyreduce angiogenesis.

The ZFPs provided include one or more zinc fingers with at least onefinger having an amino acid sequence as shown in a row of Table 3 or 4.These ZFPs include those that bind to specific sequences within variousVEGF genes. Such binding can be utilized to regulate angiogenesis andtreat ischemia and various other disorders dependent upon proper bloodflow. Tables 3 and 4 show the amino acid sequences of a large collectionof ZFPs that bind to particular target sites within different VEGFgenes. The nucleotide target site is shown in column 2 of these tables.Thus, certain ZFPs disclosed herein recognize a target site that has anucleotide sequence as specified in Tables 3 and 4. Some of these ZFPsinclude 1-6 fingers (although other ZFPs can have more fingers) andthese fingers are occupied by the amino acids shown in a row of Table 3or 4. These amino acids can occupy positions −1 to +6 of a zinc finger.

Certain of the ZFPs provided herein recognize a target site thattypically has 9 nucleotides. In general, such target sites include threetarget subsites bound by respective zinc finger components of amultifinger protein. Examples of such target sites are listed in Table3. The amino acid sequences of portions of the zinc finger componentsinvolved in recognition are shown in columns 4, 6 and 8. For someproteins that have three zinc fingers, the fingers are occupied byfirst, second and third segments of seven contiguous amino acids asshown in Table 3.

Other ZFPs that are disclosed herein bind target sequences thattypically include 18 nucleotides. These target sequences can berecognized by ZFPs that include six fingers. Examples of such ZFPs arelisted in Table 4. Hence, certain of the present ZFPs include the sixfinger ZFPs shown in Table 4, with positions −1 to +6 in each of thefingers being occupied by a segment of seven contiguous amino acids asspecified in a row of Table 4.

As indicated above, some of the ZFPs provided herein are useful inmethods for modulating angiogenesis. In some methods, the modulation ofangiogenesis comprises inhibition of new blood vessel formation. In somemethods, the modulation of angiogenesis comprises stimulation of newblood vessel formation. In some such methods the blood vessels arenonpermeable or nonhyperpermeable.

Thus, also provided are ZFPs that bind to a target site having anucleotide sequence as specified in Table 3 or 4, thereby modulatingangiogenesis when introduced into an animal having a genome comprising aVEGF gene comprising the target site. Often any of the foregoing typesof ZFPs are part of a fusion protein that includes a regulatory domain.This regulatory domain can be an activator or a repressor.

In like manner, certain of the methods for modulating angiogenesis asprovided herein involve introducing a ZFP that binds to a target sitespecified in Table 3 or 4 into an animal having a genome comprising aVEGF gene that includes the target site, with binding of the ZFP to thetarget site resulting in modulation of angiogenesis in the animal.Related methods involve contacting a target site of a nucleic acidwithin a cell with a zinc finger protein, wherein the target site has anucleotide sequence as specified in Table 3 or 4 and binding of the zincfinger protein to the target site modulates expression of the VEGF genein the cell.

The ZFPs provided herein can be used to treat a number of differentdiseases that are correlated with regulating the formation of bloodvessels. Thus, certain methods are designed to treat ischemia. Some ofthese methods involve administering a ZFP that binds to a target site asspecified in Table 3 or 4 into an animal having ischemia, wherein theZFP is administered in an amount effective to treat ischemia. The ZFPscan also be utilized, for example, in wound treatment, a variety ofsurgical applications and in promoting the growth of lymphaticendothelial cells and the activation of myelopoiesis. Other ZFPs finduse in preventing unwanted processes by repressing vessel formation.Thus, for example, ZFPs can be used in treating diabetic retinopathy,psoriasis, arthropathies and tumor growth.

By selecting certain ZFPs one can tailor the extent to which aphysiological process (e.g., angiogenesis) can be modulated and tailortreatment. This can be achieved because multiple target sites in anygiven gene can be acted upon by the ZFPs provided herein and because asingle ZFP can bind to a target site located in a plurality of genes.Thus, in some methods, a plurality of ZFPs are administered. These ZFPscan then bind to different target sites located within the same gene.Such ZFPs can in some instances have a synergistic effect. In certainmethods, the plurality of fusion proteins include different regulatorysequences. In contrast, with some of the ZFPs provided herein,administration of a single ZFP can modulate gene expression of multiplegenes because each gene includes the target site.

Also provided herein are nucleotides that encode the ZFPs disclosedherein.

Additionally, pharmaceutical compositions containing the nucleic acidsand/or ZFPs are also provided. For example, certain compositions includea nucleic acid that encodes one of the ZFPs described herein operablylinked to a regulatory sequence and a pharmaceutically acceptablecarrier or diluent, wherein the regulatory sequence allows forexpression of the nucleic acid in a cell. Protein based compositionsinclude a ZFP as disclosed herein and a pharmaceutically acceptablecarrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show an analysis of regions of enhanced chromatinaccessibility in the promoter of VEGF-A.

FIGS. 1A and 1B show analysis of constitutive accessible sites.Chromatin from the indicated human (FIG. 1A) or rat (FIG. 1B) cells waspartially digested with DNAse I in permeabilized nuclei (see“Experimental Procedures”), followed by Southern blot analysis using theindicated restriction enzymes and probes. The vertical bar representsgenomic DNA in the VEGF-A promoter region. A hooked arrow denotes thetranscription start site, and tick marks indicate units of 100 bp.Positions of restriction enzyme recognition sites are indicated in basepairs relative to the start site of VEGF-A transcription. The migrationpattern for a set of DNA standard fragments is indicated at the rightside of each gel, with the size of each fragment given in bp. Arrows areused to highlight the relationship of the observed bands to location ofaccessible chromatin regions relative to the transcription start sitesof VEGF-A. DNAse I concentrations (in Worthington Units/ml) were asfollows: HEK 293 nuclei (FIG. 1A, lanes 1-4): 0, 7.5, 15, 60; HEP3Bnuclei (FIG. 1A, lanes 5-8): 0, 7.5, 15, 30; cardiac myocyte nuclei(FIG. 1B, lanes 1-4): 0, 3.75, 7.5, 15; H9c2(2-1) nuclei (FIG. 1B, lanes5-8): 0, 15, 30, 60.

FIG. 1C shows the results of an experiment indicating the presence of anaccessible region ˜1000 base pairs upstream of the VEGF-A transcriptionstart site in primary skeletal muscle cells but not HEK 293 cells.Details as in FIGS. 1A and 1B, except that nuclei were isolated from HEK293 cells or human primary skeletal muscle cells; DNAse I concentrations(Units/ml) were as follows: HEK 293 nuclei (lanes 1-5): 0, 7.5, 15, 30,60; primary skeletal muscle cell nuclei (lanes 6-10): 0, 3.75, 7.5, 15,30.

FIG. 1D shows the results of an experiment indicating the presence of anaccessible region 500 by downstream of the VEGF-A transcription startsite in HEK 293 cells. Details as in FIGS. 1A and 1B. DNAse Iconcentrations (Units/ml) were as follows (lanes 1-5): 0, 15, 30, 60,120.

FIG. 1E shows a summary of DNAse I accessible regions observed in thesestudies. Cell types tested are indicated at left. Observation of aparticular open region in a given cell type is denoted by a an arrow.The ‘−550’ and ‘+1’ hypersensitive regions occur in all cell typestested, while the ‘−1000’ and ‘+500’ sites appear only in a subset oftested cells. A schematic representation of the VEGF-A promoter,encompassing bases −1000 to +1000 relative to the principaltranscription start site, is provided at bottom. The filled arrowdenotes the principal site of transcription initiation, while analternate start site is highlighted by an open arrow. Key regulatoryelements are also shown (HRE: hypoxia response element; binding sitesfor the SP1 and AP2 transcription factors are also shown). DNAse Iaccessible regions are indicated by gradient-shaded rectangles above themap.

FIG. 1F shows an analysis of the extent of sequence conservation betweenman, mouse and rat in the promoter region of the VEGF-A gene. Each pointin the gray profile indicates the fractional conservation of humanVEGF-A sequence in both rat and mouse within a 50 by window centered onthat point. The black profile is identical except that it indicates thefractional conservation of 5 by blocks.

FIGS. 2A-2D show the scheme for targeting construction of VEGF-Atargeted ZFPs.

FIG. 2A shows a schematic diagram depicting the structure of anindividual zinc finger with two β-sheets linked to the DNA-bindingα-helix. The diagram is based on Pavletich et al. (1991) Science 252:809-817. Oligos 1, 3 and 5 encode the β-sheet regions, and oligos 2, 4,and 6 encode the DNA-binding α-helix regions.

FIG. 2B shows the scheme for assembly of ZFP-encoding nucleic acids. Sixoverlapping oligonucleotides were annealed, gaps were filled, and theresulting duplex was amplified using a pair of externaloligonucleotides. The PCR products were then cut with Kpn I and BamHI,and the digestion product was cloned into the pMalC2 bacterialexpression vector.

FIG. 2C shows a schematic diagram of the maltose bindingprotein—VEGF-A-targeted ZFP fusions.

FIG. 2D shows a schematic representation of the human VEGF-A gene,showing the location of transcription initiation sites (hooked arrows),DNAse I-accessible regions in HEK293 cells (gradient-filled rectangles),and target site locations for the VEGF-A-targeted ZFPs (verticalrectangles). The position of the upstream-most nucleotide of each ZFPtarget site is indicated by the number below it. Numbering is relativeto the start site of transcription (+1).

FIGS. 3A-3F show an analysis of the transcriptional activationproperties of ZFPs targeted to DNAse I-accessible regions of VEGF-A.

FIG. 3A gives a schematic representation of the VEGF-A promoter reporterconstruct (top) and the endogenous VEGF-A chromosomal (bottom) targets.Coverage of portions of the endogenous promoter with white circlesindicates the presence of nucleosomes in these regions. ZFP targets areindicated by white vertical rectangles, and arrows connect each targetwith the name of its corresponding ZFP below.

FIG. 3B shows results of assay for activation of the human VEGF-Apromoter reporter by ZFP-VP16 fusions. ZFP-VP16 fusion plasmids wereco-transfected into HEK293 cells with a VEGF-A reporter constructcontaining a luciferase gene under the control of a 3.4 kbp fragment ofthe human VEGF-A promoter, and reporter activity was assayed 40 hourspost-transfection as described in “Experimental Procedures.” Aconstitutive Renilla luciferase construct was also co-transfected toserve as transfection control for normalization. The fold reporteractivation by the ZFPs was calculated based on the normalized luciferasereporter activity, in comparison with that of the control vector whichencodes a VP16-FLAG fusion lacking a ZFP domain.

FIGS. 3C and 3D show the results of assays for activation of theendogenous human VEGF-A gene by ZFP-VP16 fusions. Plasmids encodingZFP-VP16 fusions were transfected into HEK 293 cells via LipofectAMINEreagent as described in “Experimental Procedures.” The control vectorexpressed VP16-FLAG fused with green fluorescent protein (GFP) insteadof ZFP. Forty hours after transfection, the culture medium and the cellswere harvested and assayed for endogenous VEGF-A expression. FIG. 3Cshows the results of measurement of VEGF-A protein content in theculture medium, by ELISA using a human VEGF ELISA kit (R&D Systems,Minneapolis, Minn.). The VEGF-A protein production induced by the ZFPswas compared with that of the control vector, and the fold activationwas plotted. FIG. 3D shows results of assays for steady-state VEGF-AmRNA levels in transfected cells, measured by quantitative RT-PCR usingTaqman chemistry as described in “Experimental Procedures.” The levelsof VEGF-A mRNA were normalized with respect to GAPDH(glyceraldehyde-phosphate dehydrogenase) mRNA levels.

FIG. 3E shows analysis of ZFP protein content in the transfected cellsby protein immunoblotting (“Western” blotting) using an anti-FLAGantibody (Sigma, St. Louis, Mo.) which recognizes the FLAG epitope tagof the engineered ZFPs.

FIG. 3F shows the results of analysis of levels of ZFP mRNA intransfected cells, as determined by Taqman, and normalized to GAPDH mRNAlevels. The primers and probe were designed to recognize the sequenceencoding the VP16 activation domain and FLAG tag.

FIGS. 4A-4C show an analysis of the effects of ZFP fusions, targeted toaccessible and inaccessible regions of the VEGF-A gene, on a VEGF-Areporter construct and on the endogenous VEGF-A gene.

FIG. 4A provides a schematic representation of the VEGF-A promoterreporter construct (top) and the endogenous VEGF-A chromosomal (bottom)targets used in this experiment. Coverage of portions of the endogenouspromoter with white circles indicates the presence of nucleosomes inthese regions. ZFP target sites are indicated by white verticalrectangles, and arrows connect each target with the name of itscorresponding ZFP below.

FIG. 4B shows activation of a human VEGF-A promoter reporter. Theindicated ZFP-VP16 fusion plasmids were co-transfected with theVEGF-A-luciferase reporter construct as described in “ExperimentalProcedures.” The fold-activation of luciferase activity by the ZFPs wascalculated in comparison with that of a control vector encodingVP16-FLAG without a ZFP domain.

FIG. 4C shows activation of the endogenous human VEGF-A gene. Plasmidsencoding the indicated ZFP-VP16 fusions were transfected into HEK 293cells, and the amount of VEGF-A protein secreted into the culturemedium, 40 hrs after transfection, was measured by ELISA as described inthe legend to FIG. 3C. VEGF-A protein production induced by the ZFPs wascompared with that of the control vector, and the fold activation wasplotted.

FIGS. 5A-5D show activation of the endogenous human VEGF-A gene by ZFPswith different activation domains. Various ZFPs were fused with either aVP16 activation domain or the activation domain from NF-κB (p65). Allfusions also contained a C-terminal FLAG epitope tag.

FIG. 5A shows schematic diagrams of VEGF-A targeted ZFP fusionscontaining either a VP16- or a p65 activation domain (AD). NLS: nuclearlocalization sequence; ZFP: zinc finger DNA-binding domain; FLAG: Flagepitope tag.

FIG. 5B shows an analysis of ZFP protein content in transfected cells,analyzed by western blotting using anti-FLAG antibody. Identity of thetransfected ZFP domain is indicated above each lane of the gel.

FIG. 5C shows VEGF-A protein content in the culture medium oftransfected cells, measured by ELISA. Results from transfection of ZFPfusions comprising a VP16 activation domain are represented by openbars; results from transfection of ZFP fusions comprising a p65activation domain are represented by filled bars.

FIG. 5D shows measurement of steady-state VEGF-A mRNA levels in thetransfected cells by Taqman. Results from transfection of ZFP fusionscomprising a VP16 activation domain are represented by open bars;results from transfection of ZFP fusions comprising a p65 activationdomain are represented by filled bars.

FIGS. 6A-6D show an analysis of cooperativity, in VEGF-A geneactivation, between ZFPs with different activation domains. Plasmidscontaining different ZFP-activation domain fusions were cotransfected ata 1:1 ratio into HEK 293 cells, and endogenous VEGF-A activation wasmeasured 40 hours after transfection.

FIG. 6A shows the scheme of the experiment.

FIG. 6B shows analysis of VEGF-A protein content in the culture medium,assayed by ELISA.

FIG. 6C shows analysis of VEGF-A mRNA levels in transfected cells,measured by Taqman.

FIG. 6D shows analysis of ZFP protein contents in the transfected cells,by western blotting using anti-FLAG antibody.

FIGS. 7A-7D show a comparison of the activation of the endogenous humanVEGF-A gene by ZFP VZ+434b and by hypoxia. HEK 293 cells weretransfected with plasmids encoding the ZFP VZ+434b fused with VP16 orp65, transfected with a control vector expressing no ZFP, or exposed tohypoxic conditions (0.5% O2) in a hypoxic incubator for 24 hours.Endogenous VEGF-A gene activation was measured as described in FIG. 6and “Experimental Procedures.”

FIG. 7A show analysis of VEGF-A protein content in the culture medium,measured by ELISA.

FIG. 7B shows analysis of steady-state VEGF-A mRNA levels, measured byTaqman, and normalized to levels of 18S RNA.

FIG. 7C shows analysis of VEGF-A mRNA by RNA blot (“Northern”)hybridization using a 32P-labeled VEGF165 riboprobe.

FIG. 7D shows analysis of VEGF-A splice variants by RT-PCR and Southernhybridization as described in “Experimental Procedures.”

FIG. 8 shows immunoblot analyses of VEGF in ZFP-injected andcontrol-injected mouse quadriceps muscle. Positions of the VEGF monomerand VEGF dimer are indicated.

FIG. 9A-9F show micrographs of H&E-stained thin sections of wound tissuefrom a mouse that had been injected with a plasmid encoding a ZFP-VP16fusion. FIGS. 9A and 9B are low-power magnification; FIGS. 9C-9F arehigh-power. FIGS. 9A, 9C and 9E show sections of wound tissue that wasinjected with a control plasmid. FIGS. 9B, 9D and 9F show sections ofwound tissue that was injected with a plasmid encoding a ZFP-VP16fusion.

FIG. 10 shows levels of human VEGF-A protein, detected by ELISA, in HEK293 cells transfected with different ZFP-encoding plasmids. See Tables2-4 for the identities of the target sites and ZFP binding domains.

FIG. 11 shows levels of human VEGF-A mRNA, detected by real-time PCR

(Taqman), in HEK 293 cells transfected with different ZFP-encodingplasmids. See Tables 3 and 4 for the identities of the ZFP bindingdomains. The lower portion of the figure shows immunoblot analysis ofZFP expression in transfected cells, using an anti-FLAG antibody.

FIG. 12 shows an analysis of reporter gene activity in HEK 293 cellsthat were co-transfected with a plasmid encoding a luciferase reportergene under the transcriptional control of a VEGF promoter and differentZFP-encoding plasmids. Fold-induction of luciferase activity ispresented relative to a control plasmid in which ZFP-encoding sequenceswere replaced by sequences encoding green fluorescent protein.

FIG. 13 shows levels of human VEGF-A protein, detected by ELISA, in HEK293 cells transfected with different ZFP-encoding plasmids. See Tables2-4 for the identities of the target sites and ZFP binding domains.

FIGS. 14A-14B show analysis of VEGF-A and VEGF-C mRNA in cells that hadbeen transfected with different plasmids encoding ZFP-VP 16 fusions.VEGF mRNA levels were analyzed and normalized with respect to GAPDH mRNAlevels as described in Example 1. FIG. 14A shows analysis of VEGF-A mRNAlevels; FIG. 14B shows analysis of VEGF-C mRNA levels. The name of thetransfected ZFP binding domain (see Table 2) and the approximatelocation of its target site are indicated along the abscissa.

FIGS. 15A-D illustrate the targeting, design, and results of DNA bindinganalyses conducted with zinc finger proteins targeted to the mouseVEGF-A locus.

FIG. 15A depicts the results of mapping of DNase 1 accessible regions inthe mouse VEGF-A promoter region. Nuclei from the indicated cell lineswere partially digested with DNase I (see “Experimental Procedures”),followed by Southern blot analysis using Bgl 1 and the indicated probe.The vertical bar represents the promoter region of the VEGF-A locus. Ahooked arrow denotes the transcription start site, and tick marksindicate units of 100 bp. The migration pattern for a set of DNAstandard fragments is indicated at the right side of the gel, with thesize of each fragment given in base pairs. Arrows are used to highlightthe relationship of observed bands to location of accessible chromatinregions relative to the transcription start site of VEGF-A. DNAse Iconcentrations (U/ml) in lanes 1-6 were as follows: 0, 32, 64, 0, 8, and16, respectively.

FIG. 15B shows the location of ZFP target sites used for one set ofbinding studies. A schematic representation of the human VEGF-A gene isprovided, showing the location of the principal (filled arrow) and areported alternate (open arrow) transcription initiation site, and theDNAse I accessible regions (gradient-filled rectangles) determined inthese studies. ZFP target locations are indicated by vertical openrectangles, and the position of the 5′-most nucleotide of each ZFPtarget is indicated by the number below it. Numbering is relative to thestart site of transcription.

FIG. 15C lists ZFP target sequences (SEQ ID NOS:207, 144, and 240,respectively) and finger designs (SEQ ID NOS:239, 238, 122, 57, 159, 35,64, 85, 36, 112, 66, and 54, respectively). ZFPs are named according totarget site location and the suffix mVZ (for mouse VEGF-A ZFP). Fingerdesigns indicate the identity of amino acid residues at positions −1 to+6 of the alpha helix of each finger.

FIG. 15D shows gel-shift assays of binding affinity. A three-folddilution series of each protein was tested for binding to its DNA target(SEQ ID NOS:207, 144, 240, and 141, respectively), with the highestconcentration in lane 10 and the lowest concentration in lane 2. Lane 1contains probe alone. Apparent Kd's, derived from the average of 3 suchstudies, are indicated at right. For mVZ+426 and mVZ+509, Kd's areprovided as upper bounds (<0.01 nM), since the use of 0.01 nM of probehas probably led to an underestimate of the affinity of these proteins.

FIGS. 16A-16C depict ZFP-mediated activation of the mouse VEGF-A locusin C127I cells. Cells were transduced with retroviral vectors expressingeach ZFP transcriptional activator or a control protein in which greenfluorescent protein (GFP) was substituted for the ZFP DNA-bindingdomain. After selection for drug resistance, the resultant cellpopulation was assayed for expression of VEGF-A by TaqMan or ELISA.

FIG. 16A is a schematic representation of the ZFP transcriptionalactivators used in these studies. ‘NLS’, ‘ZFP,’ VP16′ and ‘FLAG’indicate the relative locations of, respectively, a nuclear localizationsignal, a ZFP binding domain, VP 16 activation domain, and FLAG tag. See“Experimental Procedures” for a detailed description of the constructs.

FIG. 16B shows the measurement of ZFP-mediated activation of the VEGF-Alocus in C127 I cells by TaqMan™ analysis. Expression of VEGF-A mRNA isnormalized to GAPDH mRNA level.

FIG. 16C shows results of ZFP-mediated activation of VEGF-A locus byquantitating the level of secreted VEGF-A protein via ELISA (R&Dsystems).

FIGS. 17A-17D show Western blots that demonstrate induction of VEGF Aexpression by ZFPs.

FIG. 17A shows the results of a Western blot that demonstratesexpression of a ZFP fusion protein in cultured smooth muscle cellstransduced with a recombinant adenovirus construct encoding a fusionprotein comprising an NLS, ZFP VOP 30A, a VP16 activation domain and aFLAG epitope. Both anti-VP16 and anti-FLAG antibodies were used, asindicated. Cells transduced with a recombinant adenovirus encoding greenfluorescent protein were used as control.

FIG. 17B presents a Western blot demonstrating a marked increase inVEGF-A expression in the hindlimb adductor muscle of CD-1 mice wheninjected with recombinant adenovirus encoding VOP 30A as compared to anadenovirus encoding green fluorescent protein (GFP) as control.Duplicate samples are shown.

FIG. 17C is a Western blot showing induction of VEGF-A proteinexpression following injection of adeno-MVG (see Example 6) into mouseskeletal muscle relative to a control injection of an adenovirusencoding green fluorescent protein (GFP). Two different mice wereinjected with MVG and two separate mice with control as indicated.Levels of actin were also determined on the immunoblot as a loadingcontrol.

FIG. 17D shows a Western blot illustrating induction of VEGF-A in ratskeletal muscle after injection of plasmids encoding the VEGF-Amodulating ZFPs VOP 30A, VOP 32B, BVO12A, BV014A as compared to acontrol plasmid encoding a ZFP backbone but lacking the DNA recognitiondomain.

FIGS. 18A-E show results demonstrating induction of angiogenesis usingan established model system.

FIGS. 18A and 18B are photographs of vascularization in mouse earsfollowing injection with a recombinant adenovirus encoding either greenfluorescent protein (GFP) as control (FIG. 18A) or a ZFP VOP 30A (FIG.18B).

FIGS. 18C and 18D show similar photographs as those presented in FIGS.18A and 18B but with an adenovirus encoding either green fluorescentprotein (GFP) (FIG. 18C) or ZFP VOP 32 B (FIG. 18D).

FIG. 18E is a chart of the number of blood vessels as determined usingimmunostaining techniques (see Experimental Procedures section infra) ateither three or six days after injection of a mouse ear with adenovirusof the type described in FIGS. 18C and 18D. The chart demonstratesincreased vascularity and is consistent with the results shown in FIGS.18A and 18B.

FIGS. 19A-19C present results showing that cutaneous wound healing isaccelerated by VEGF-A regulating ZFPs. Bilateral cutaneous wounds werecreated in the backs of CD-1 mice by excising a 5 mm circle of skin. Atthe time of wounding, adenovirus encoding a VEGF-A regulating zincfinger protein or a control adenovirus encoding green fluorescentprotein (GFP) was applied topically to the wound. Wounds were excisedwhole 5 days later, the tissue was fixed and paraffin embedded forhistologic and immunohistologic analysis.

FIG. 19A illustrates the extent of reepithelialization followingwounding and subsequent treatment by adenovirus encoding greenfluorescent protein (GFP).

FIG. 19B illustrates how treatment with the adenovirus MVG augments thedegree of reepithelialization noted at day 5 post-wounding. The arrowsdenote the leading edge of keratinocyte ingrowth into the wound. As thisfigure illustrates, the distance between the edges of keratinocyteingrowth is decreased by VEGF-ZFP treatment; thus reepithelialization isaugmented.

FIG. 19C is a chart summarizing the distance between leading edges ofkeratinocyte ingrowth in wounds treated with either adenovirus encodinga ZFP (dark shading) or green fluorescent protein (GFP) (light shading)and shows a significant reduction in distance for treatment with theVEGF-ZFP treatment as compared to the control.

FIGS. 20A and 20B show that wound reepithelialization is augmented bytreatment with the adenovirus MVG. The lower arrowhead in eachphotograph marks the wound edge and the upper arrow marks the extent ofkeratinocyte ingrowth at day 5 post-wounding with a recombinantadenoviruses encoding MVG (FIG. 20B) or green fluorescent protein (GFP)as control (FIG. 20B).

FIGS. 21A-21C show that treatment of cutaneous wounds by topicalapplication of recombinant adenovirus encoding a VEGF-A regulating ZFP(MVG) increases vascularity (FIG. 21A) as compared to an adenovirusencoding green fluorescent protein (GFP) (FIG. 21B). The presence ofvessels is visualized with an immunostain containing antibodies specificfor endothelial cells. FIG. 21C is a chart that summarizes vessel countsperformed with digitally captured images when wounds were treated withadenovirus encoding a ZFP (dark box) or green fluorescent protein (GFP)(light box).

FIG. 22 shows angiogenesis stimulated by MVZ+509 (top and middle rightpanels) does not produce a hyperpermeable neovasculature as determinedby Evans blue dye extravasation (bottom right). The neovasculatureinduced by VEGF 164 adenovirus transduction (left panels) exhibitspontaneous hemorrhage and Evans blue extravasation.

DETAILED DESCRIPTION I. Definitions

The practice of conventional techniques in molecular biology,biochemistry, cell culture, recombinant DNA, and related fields arewell-known to those of skill in the art and are discussed, for example,in the following literature references: Sambrook et al. MOLECULARCLONING: A LABORATORY MANUAL, Second edition, Cold Spring HarborLaboratory Press, 1989; Ausubel et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; theseries METHODS IN ENZYMOLOGY, Academic Press, San Diego; and METHODS INMOLECULAR BIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.)Humana Press, Totowa, 1999, all of which are incorporated by referencein their entireties.

The term “zinc finger protein” or “ZFP” refers to a protein having DNAbinding domains that are stabilized by zinc. The individual DNA bindingdomains are typically referred to as “fingers” A ZFP has least onefinger, typically two, three, four, five, six or more fingers. Eachfinger binds from two to four base pairs of DNA, typically three or fourbase pairs of DNA. A ZFP binds to a nucleic acid sequence called atarget site or target segment. Each finger typically comprises anapproximately 30 amino acid, zinc-chelating, DNA-binding subdomain. Anexemplary motif characterizing one class of these proteins (C2H2 class)is -Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His (where X is any amino acid) (SEQID NO:208). Additional classes of zinc finger proteins are known and areuseful in the practice of the methods, and in the manufacture and use ofthe compositions disclosed herein (see, e.g., Rhodes et al. (1993)Scientific American 268:56-65). Studies have demonstrated that a singlezinc finger of this class consists of an alpha helix containing the twoinvariant histidine residues coordinated with zinc along with the twocysteine residues of a single beta turn (see, e.g., Berg & Shi, Science271:1081-1085 (1996)).

A “target site” is the nucleic acid sequence recognized by a ZFP. Asingle target site typically has about four to about ten base pairs.Typically, a two-fingered ZFP recognizes a four to seven base pairtarget site, a three-fingered ZFP recognizes a six to ten base pairtarget site, and a six fingered ZFP recognizes two adjacent nine to tenbase pair target sites.

A “target subsite” or “subsite” is the portion of a DNA target site thatis bound by a single zinc finger, excluding cross-strand interactions.Thus, in the absence of cross-strand interactions, a subsite isgenerally three nucleotides in length. In cases in which a cross-strandinteraction occurs (i.e., a “D-able subsite,” see co-owned WO 00/42219)a subsite is four nucleotides in length and overlaps with another 3- or4-nucleotide subsite.

“Kd” refers to the dissociation constant for a binding molecule, i.e.,the concentration of a compound (e.g., a zinc finger protein) that giveshalf maximal binding of the compound to its target (i.e., half of thecompound molecules are bound to the target) under given conditions(i.e., when [target]<<Kd), as measured using a given assay system (see,e.g., U.S. Pat. No. 5,789,538). The assay system used to measure the Kdshould be chosen so that it gives the most accurate measure of theactual Kd of the ZFP. Any assay system can be used, as long is it givesan accurate measurement of the actual Kd of the ZFP. In one embodiment,the Kd for a ZFP is measured using an electrophoretic mobility shiftassay (“EMSA”). Unless an adjustment is made for ZFP purity or activity,the Kd calculations may result in an overestimate of the true Kd of agiven ZFP. Preferably, the Kd of a ZFP used to modulate transcription ofa gene is less than about 100 nM, more preferably less than about 75 nM,more preferably less than about 50 nM, most preferably less than about25 nM.

The term “VEGF gene” refers generally to any member of the VEGF familyof genes as described supra or collection of genes from the VEGF familyhaving a native VEGF nucleotide sequence, as well as variants andmodified forms regardless of origin or mode of preparation. The VEGFgenes can be from any source. Typically, the VEGF genes refer to VEGFgenes in mammals, particularly humans. A VEGF gene having a nativenucleotide sequence is a gene having the same nucleotide sequence as aVEGF gene as obtained from nature (i.e., a naturally occurring VEGFgene). More specifically, the term includes VEGF-A (including theisoforms VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189, and VEGF-A206);VEGF-B (including the isoforms VEGF-B167, and VEGF-B186); VEGF-C;VEGF-D; VEGF-E (various VEGF-like proteins from orf virus strains asdescribed in the Background section); VEGF-H; VEGF-R; VEGF-X; VEGF-138;and P1GF (both P1GF-1 and P1GF-2). The term also includes variants ofspecific isoforms. For example, the term includes not only the isoformVEGF-145, but also VEGF-145-I, VEGF-145-II, and VEGF-145-III. The termalso encompasses allelic variants, other isoforms resulting fromalternative exon splicing, forms that are functionally equivalent tonative sequences, and nucleic acids that are substantially identical toa native VEGF gene. More specifically, the term encompasses thefollowing VEGF genes.

VEGF (VEGF-A) has been described by a number of researchers (see, e.g.,Leung, et al. (1989) Science 246:1306-1309; Keck, et al. (1989) Science246:1309-1312; and Conn et al. (1990) Proc. Natl. Acad. Sci. USA87:2628-2632, each of which is incorporated herein in its entirety). TheVEGF-A189 isoform is disclosed in U.S. Pat. No. 5,240,848, the VEGF-A121isoform in U.S. Pat. Nos. 5,194,596 and 5,219,739; and the VEGF-A165isoform in U.S. Pat. No. 5,332,671, each of which is incorporated byreference in its entirety.

VEGF-B is described in PCT Publication WO 96/26736, in U.S. Pat. No.5,840,693, U.S. Pat. No. 5,607,918, and U.S. Pat. No. 5,928,939, each ofwhich is incorporated herein in its entirety. See also, PCT PublicationsWO 96/27007 and WO 00/09148, and Olofsson et al. (1996) Proc. Natl.Acad. Sci. USA 93:2576-2581, each of which is incorporated herein byreference in its entirety.

VEGF-C is disclosed by Joukov et al., (1996) EMBO J. 15:290-298, and Leeet al. (1996) Proc. Natl. Acad. Sci. USA 93:1988-1992, as well as inU.S. Pat. No. 5,935,820; and U.S. Pat. No. 6,130,071, each of which isincorporated herein in its entirety. See also U.S. Pat. Nos. 5,776,755and 5,932,540, as well as PCT Publications WO 95/24473; WO 96/39515; WO97/05250; WO 97/09427; WO 97/17442; WO 98/33917; and WO 99/46364, eachof which is incorporated herein by reference in its entirety. Otherforms are discussed in EP 0 476 983 B1; U.S. Pat. Nos. 5,994,300 and6,040,157; and PCT publication WO 0.00/45835, each of which areincorporated by reference in its entirety.

VEGF-D is described in PCT Publications WO 98/07832, WO 98/24811; and WO99/33485. It is further described by Achen et al. (1988) Proc. Natl.Acad. Sci. USA 95:548-553, each of the foregoing being incorporatedherein in its entirety. See also EP 0 935 001 A1, which is incorporatedherein in its entirety.

The term also includes the various viral forms described in theBackground section that are collectively referred to herein as VEGF-E.Such viral VEGF-like genes include the gene isolated from the orf viralstrain NZ2 that is referred to in the literature variously as OV NZ2,ORFV2-VEGF, OV-VEGF2 and VEGF-ENZ2A (see, e.g., Lyttle, D. J. et al.(1994) J. Virology 68:84-92; and PCT Publication WO 00/25805). Alsoincluded are the gene identified in strain NZ7 (called NZ7, OV-VEGF7,VEGF-E and VEGF-ENZ7). This gene is discussed in Lyttle, D. J. et al.(1994) J. Virology 68:84-92; and Ogawa, S. et al. (1998) J. Biol. Chem.273:31273-31282. Also included is the NZ10 gene which is disclosed inPCT Publication WO 00/25805. The gene from orf viral strain D1701 isalso included (see, e.g., Meyer et al. (1999) EMBO J. 18:363-74, whichis incorporated herein by reference in its entirety). Another viralVEGF-like protein from para-poxvirus has been disclosed in PCTPublication WO 99/50290.

The term further includes the mammalian VEGF-like protein that has alsobeen referred to as VEGF-E (see, e.g., WO 99/4767)

The term also includes the gene called PDGFNEGF-Like Growth Factor H, orsimply VEGF-H that is discussed in PCT Publication WO 00/44903(incorporated by reference in its entirety). VEGF-R (see, e.g., PCTPublication WO 99/37671, incorporated by reference in its entirety) andVEGF-X (see, e.g., PCT publication WO 00/37641, incorporated herein byreference in its entirety) are also included, as is the recentlyidentified VEGF-138 gene. The various isoforms (P1GF1 and P1GF2) of thePlacenta Growth Factor, P1GF, are further included in the term. Methodsfor isolating and characterizing this gene and the protein it encodesare set forth in Maglione et al. (1991) Proc. Natl. Acad. Sci. USA88:9267-9271, which is incorporated herein by reference in its entirety.See also, Birkenhager, R. (1996) Biochem. J. 316:703-707; Kao, Y. (1997)Biochem. Biophys. Res. Commun. 235:493-498; Kao, Y. (1996) J. Biol.Chem. 271:3154-3162; and Klauss, M. (1996) J. Biol. Chem.271:17629-17634, each of which is incorporated by reference in itsentirety.

As indicated supra, the term VEGF gene includes nucleic acids that aresubstantially identical to a native sequence VEGF gene. The terms“identical” or percent “identity,” in the context of two or more nucleicacids or polypeptides, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of nucleotides or aminoacid residues that are the same, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm suchas those described below for example, or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 75%, preferably at least 85%, more preferably atleast 90%, 95% or higher or any integral value therebetween nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using a sequence comparison algorithm suchas those described below for example, or by visual inspection.Preferably, the substantial identity exists over a region of thesequences that is at least about 10, preferably about 20, morepreferable about 40-60 residues in length or any integral valuetherebetween, preferably over a longer region than 60-80 residues, morepreferably at least about 90-100 residues, and most preferably thesequences are substantially identical over the full length of thesequences being compared, such as the coding region of a nucleotidesequence for example.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection [see generally,Current Protocols in Molecular Biology, (Ausubel, F. M. et al., eds.)John Wiley & Sons, Inc., New York (1987-1999, including supplements suchas supplement 46 (April 1999)]. Use of these programs to conductsequence comparisons are typically conducted using the defaultparameters specific for each program.

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. This is referred to as theneighborhood word score threshold (Altschul et al, supra.). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. For determining sequence similarity the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent conditions. “Hybridizes substantially” refers to complementaryhybridization between a probe nucleic acid and a target nucleic acid andembraces minor mismatches that can be accommodated by reducing thestringency of the hybridization media to achieve the desired detectionof the target polynucleotide sequence. The phrase “hybridizingspecifically to”, refers to the binding, duplexing, or hybridizing of amolecule only to a particular nucleotide sequence under stringentconditions when that sequence is present in a complex mixture (e.g.,total cellular) DNA or RNA.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below.

“Conservatively modified variations” of a particular polynucleotidesequence refers to those polynucleotides that encode identical oressentially identical amino acid sequences, or where the polynucleotidedoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every polynucleotidesequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

A polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. A “conservative substitution,” whendescribing a protein, refers to a change in the amino acid compositionof the protein that does not substantially alter the protein's activity.Thus, “conservatively modified variations” of a particular amino acidsequence refers to amino acid substitutions of those amino acids thatare not critical for protein activity or substitution of amino acidswith other amino acids having similar properties (e.g., acidic, basic,positively or negatively charged, polar or non-polar, etc.) such thatthe substitutions of even critical amino acids do not substantiallyalter activity. Conservative substitution tables providing functionallysimilar amino acids are well-known in the art. See, e.g., Creighton(1984) Proteins, W.H. Freeman and Company. In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence are also “conservatively modified variations.”

The term “VEGF protein” refers to a protein encoded by a VEGF gene andincludes functional equivalents of such proteins.

A “functional fragment” or “functional equivalent” of a protein,polypeptide or nucleic acid is a protein, polypeptide or nucleic acidwhose sequence is not identical to the full-length protein, polypeptideor nucleic acid, yet retains the same function as the full-lengthprotein, polypeptide or nucleic acid. A functional fragment can possessmore, fewer, or the same number of residues as the corresponding nativemolecule, and/or can contain one or more amino acid or nucleotidesubstitutions. Methods for determining the function of a nucleic acid(e.g., coding function, ability to hybridize to another nucleic acid,binding to a regulatory molecule) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. See Ausubel et al., supra. The ability of aprotein to interact with another protein can be determined, for example,by co-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form. Forthe purposes of the present disclosure, these terms are not to beconstrued as limiting with respect to the length of a polymer. The termscan encompass known analogues of natural nucleotides, as well asnucleotides that are modified in the base, sugar and/or phosphatemoieties. In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T. Thus, the term polynucleotide sequence is the alphabeticalrepresentation of a polynucleotide molecule. This alphabeticalrepresentation can be input into databases in a computer having acentral processing unit and used for bioinformatics applications such asfunctional genomics and homology searching. The terms additionallyencompass nucleic acids containing known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Examples of such analogs include,without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,and peptide-nucleic acids (PNAs). The nucleotide sequences are displayedherein in the conventional 5′-3′ orientation.

Chromatin is the nucleoprotein structure comprising the cellular genome.“Cellular chromatin” comprises nucleic acid, primarily DNA, and protein,including histones and non-histone chromosomal proteins. The majority ofeukaryotic cellular chromatin exists in the form of nucleosomes, whereina nucleosome core comprises approximately 150 base pairs of DNAassociated with an octamer comprising two each of histones H2A, H2B, H3and H4; and linker DNA (of variable length depending on the organism)extends between nucleosome cores. A molecule of histone H1 is generallyassociated with the linker DNA. For the purposes of the presentdisclosure, the term “chromatin” is meant to encompass all types ofcellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome” is a chromatin complex comprising all or a portion of thegenome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “exogenous molecule” is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. Normal presence in the cell is determinedwith respect to the particular developmental stage and environmentalconditions of the cell. Thus, for example, a molecule that is presentonly during embryonic development of muscle is an exogenous moleculewith respect to an adult muscle cell. An exogenous molecule cancomprise, for example, a functioning version of a malfunctioningendogenous molecule or a malfunctioning version of anormally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., protein or nucleic acid (i.e., an exogenous gene),providing it has a sequence that is different from an endogenousmolecule. Methods for the introduction of exogenous molecules into cellsare known to those of skill in the art and include, but are not limitedto, lipid-mediated transfer (i.e., liposomes, including neutral andcationic lipids), electroporation, direct injection, cell fusion,particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous molecule” is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions.

The phrase “adjacent to a transcription initiation site” refers to atarget site that is within about 50 bases either upstream or downstreamof a transcription initiation site. “Upstream” of a transcriptioninitiation site refers to a target site that is more than about 50 bases5′ of the transcription initiation site (i.e., in the non-transcribedregion of the gene). “Downstream” of a transcription initiation siterefers to a target site that is more than about 50 bases 3′ of thetranscription initiation site.

A “fusion molecule” is a molecule in which two or more subunit moleculesare linked, typically covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion polypeptides (for example, a fusion between aZFP DNA-binding domain and a transcriptional activation domain) andfusion nucleic acids (for example, a nucleic acid encoding the fusionpolypeptide described supra). Examples of the second type of fusionmolecule include, but are not limited to, a fusion between atriplex-forming nucleic acid and a polypeptide, and a fusion between aminor groove binder and a nucleic acid.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Gene activation” refers to any process that results in an increase inproduction of a gene product. A gene product can be either RNA(including, but not limited to, mRNA, rRNA, tRNA, and structural RNA) orprotein. Accordingly, gene activation includes those processes thatincrease transcription of a gene and/or translation of a mRNA. Examplesof gene activation processes that increase transcription include, butare not limited to, those that facilitate formation of a transcriptioninitiation complex, those that increase transcription initiation rate,those that increase transcription elongation rate, those that increaseprocessivity of transcription and those that relieve transcriptionalrepression (by, for example, blocking the binding of a transcriptionalrepressor). Gene activation can constitute, for example, inhibition ofrepression as well as stimulation of expression above an existing level.Examples of gene activation processes which increase translation includethose that increase translational initiation, those that increasetranslational elongation and those that increase mRNA stability. Ingeneral, gene activation comprises any detectable increase in theproduction of a gene product, in some instances an increase inproduction of a gene product by about 2-fold, in other instances fromabout 2- to about 5-fold or any integer therebetween, in still otherinstances between about 5- and about 10-fold or any integertherebetween, in yet other instances between about 10- and about 20-foldor any integer therebetween, sometimes between about 20- and about50-fold or any integer therebetween, in other instances between about50- and about 100-fold or any integer therebetween, and in yet otherinstances between 100-fold or more.

“Gene repression” and “inhibition of gene expression” refer to anyprocess which results in a decrease in production of a gene product. Agene product can be either RNA (including, but not limited to, mRNA,rRNA, tRNA, and structural RNA) or protein. Accordingly, gene repressionincludes those processes which decrease transcription of a gene and/ortranslation of a mRNA. Examples of gene repression processes whichdecrease transcription include, but are not limited to, those whichinhibit formation of a transcription initiation complex, those whichdecrease transcription initiation rate, those which decreasetranscription elongation rate, those which decrease processivity oftranscription and those which antagonize transcriptional activation (by,for example, blocking the binding of a transcriptional activator). Generepression can constitute, for example, prevention of activation as wellas inhibition of expression below an existing level. Examples of generepression processes which decrease translation include those whichdecrease translational initiation, those which decrease translationalelongation and those which decrease mRNA stability. Transcriptionalrepression includes both reversible and irreversible inactivation ofgene transcription. In general, gene repression comprises any detectabledecrease in the production of a gene product, in some instances adecrease in production of a gene product by about 2-fold, in otherinstances from about 2- to about 5-fold or any integer therebetween, inyet other instances between about 5- and about 10-fold or any integertherebetween, in still other instances between about 10- and about20-fold or any integer therebetween, sometimes between about 20- andabout 50-fold or any integer therebetween, in other instances betweenabout 50- and about 100-fold or any integer therebetween, in still otherinstances 100-fold or more. In yet other instances, gene repressionresults in complete inhibition of gene expression, such that no geneproduct is detectable.

“Modulation” refers to a change in the level or magnitude of an activityor process. The change can be either an increase or a decrease. Forexample, modulation of gene expression includes both gene activation andgene repression. Modulation can be assayed by determining any parameterthat is indirectly or directly affected by the expression of the targetgene. Such parameters include, e.g., changes in RNA or protein levels,changes in protein activity, changes in product levels, changes indownstream gene expression, changes in reporter gene transcription(luciferase, CAT, β-galactosidase, β-glucuronidase, green fluorescentprotein (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964(1997)); changes in signal transduction, phosphorylation anddephosphorylation, receptor-ligand interactions, second messengerconcentrations (e.g., cGMP, cAMP, IP3, and Ca2+), cell growth, andneovascularization. These assays can be in vitro, in vivo, and ex vivo.Such functional effects can be measured by any means known to thoseskilled in the art, e.g., measurement of RNA or protein levels,measurement of RNA stability, identification of downstream or reportergene expression, e.g., via chemiluminescence, fluorescence, colorimetricreactions, antibody binding, inducible markers, ligand binding assays;changes in intracellular second messengers such as cGMP and inositoltriphosphate (IP3); changes in intracellular calcium levels; cytokinerelease, and the like.

A “regulatory domain” or “functional domain” refers to a protein or aprotein domain that has transcriptional modulation activity whentethered to a DNA binding domain, i.e., a ZFP. Typically, a regulatorydomain is covalently or non-covalently linked to a ZFP (e.g., to form afusion molecule) to effect transcription modulation. Regulatory domainscan be activation domains or repression domains. Activation domainsinclude, but are not limited to, VP16, VP64 and the p65 subunit ofnuclear factor Kappa-B. Repression domains include, but are not limitedto, KRAB MBD2B and v-ErbA. Additional regulatory domains include, e.g.,transcription factors and co-factors (e.g., MAD, ERD, SID, early growthresponse factor 1, and nuclear hormone receptors), endonucleases,integrases, recombinases, methyltransferases, histoneacetyltransferases, histone deacetylases etc. Activators and repressorsinclude co-activators and co-repressors (see, e.g., Utley et al., Nature394:498-502 (1998)). Alternatively, a ZFP can act alone, without aregulatory domain, to effect transcription modulation.

The term “operably linked” or “operatively linked” is used withreference to a juxtaposition of two or more components (such as sequenceelements), in which the components are arranged such that bothcomponents function normally and allow the possibility that at least oneof the components can mediate a function that is exerted upon at leastone of the other components. By way of illustration, a transcriptionalregulatory sequence, such as a promoter, is operatively linked to acoding sequence if the transcriptional regulatory sequence controls thelevel of transcription of the coding sequence in response to thepresence or absence of one or more transcriptional regulatory factors.An operatively linked transcriptional regulatory sequence is generallyjoined in cis with a coding sequence, but need not be directly adjacentto it. For example, an enhancer can constitute a transcriptionalregulatory sequence that is operatively-linked to a coding sequence,even though they are not contiguous.

With respect to fusion polypeptides, the term “operably linked” or“operatively linked” can refer to the fact that each of the componentsperforms the same function in linkage to the other component as it wouldif it were not so linked. For example, with respect to a fusionpolypeptide in which a ZFP DNA-binding domain is fused to atranscriptional activation domain (or functional fragment thereof), theZFP DNA-binding domain and the transcriptional activation domain (orfunctional fragment thereof) are in operative linkage if, in the fusionpolypeptide, the ZFP DNA-binding domain portion is able to bind itstarget site and/or its binding site, while the transcriptionalactivation domain (or functional fragment thereof) is able to activatetranscription.

The term “recombinant,” when used with reference to a cell, indicatesthat the cell replicates an exogenous nucleic acid, or expresses apeptide or protein encoded by an exogenous nucleic acid. Recombinantcells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also containgenes found in the native form of the cell wherein the genes aremodified and re-introduced into the cell by artificial means. The termalso encompasses cells that contain a nucleic acid endogenous to thecell that has been modified without removing the nucleic acid from thecell; such modifications include those obtained by gene replacement,site-specific mutation, and related techniques.

A “recombinant expression cassette,” “expression cassette” or“expression construct” is a nucleic acid construct, generatedrecombinantly or synthetically, that has control elements that arecapable of effecting expression of a structural gene that is operativelylinked to the control elements in hosts compatible with such sequences.Expression cassettes include at least promoters and optionally,transcription termination signals. Typically, the recombinant expressioncassette includes at least a nucleic acid to be transcribed (e.g., anucleic acid encoding a desired polypeptide) and a promoter. Additionalfactors necessary or helpful in effecting expression can also be used asdescribed herein. For example, an expression cassette can also includenucleotide sequences that encode a signal sequence that directssecretion of an expressed protein from the host cell. Transcriptiontermination signals, enhancers, and other nucleic acid sequences thatinfluence gene expression, can also be included in an expressioncassette.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription. As used herein, a promoter typically includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of certain RNA polymerase II type promoters, a TATAelement, CCAAT box, SP-1 site, etc. As used herein, a promoter alsooptionally includes distal enhancer or repressor elements, which can belocated as much as several thousand base pairs from the start site oftranscription. The promoters often have an element that is responsive totransactivation by a DNA-binding moiety such as a polypeptide, e.g., anuclear receptor, Gal4, the lac repressor and the like.

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under certain environmental or developmentalconditions.

A “weak promoter” refers to a promoter having about the same activity asa wild type herpes simplex virus (“HSV”) thymidine kinase (“tk”)promoter or a mutated HSV tk promoter, as described in Eisenberg &McKnight, Mol. Cell. Biol. 5:1940-1947 (1985).

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell, and optionally integration or replication of the expressionvector in a host cell. The expression vector can be part of a plasmid,virus, or nucleic acid fragment, of viral or non-viral origin.Typically, the expression vector includes an “expression cassette,”which comprises a nucleic acid to be transcribed operably linked to apromoter. The term expression vector also encompasses naked DNA operablylinked to a promoter.

By “host cell” is meant a cell that contains an expression vector ornucleic acid, either of which optionally encodes a ZFP or a ZFP fusionprotein. The host cell typically supports the replication or expressionof the expression vector. Host cells can be prokaryotic cells such as,for example, E. coli, or eukaryotic cells such as yeast, fungal,protozoal, higher plant, insect, or amphibian cells, or mammalian cellssuch as CHO, HeLa, 293, COS-1, and the like, e.g., cultured cells (invitro), explants and primary cultures (in vitro and ex vivo), and cellsin vivo.

The term “naturally occurring,” as applied to an object, means that theobject can be found in nature, as distinct from being artificiallyproduced by humans.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins. Thepolypeptide sequences are displayed herein in the conventionalN-terminal to C-terminal orientation.

A “subsequence” or “segment” when used in reference to a nucleic acid orpolypeptide refers to a sequence of nucleotides or amino acids thatcomprise a part of a longer sequence of nucleotides or amino acids(e.g., a polypeptide), respectively.

The term “ischemia” broadly refers to a condition of localized anemiadue to an inadequate blood supply to the particular area (e.g., a tissueor organ), usually due to a blockage. Ischemia can be associated with avariety of diseases that involve a blockage of blood flow such ascoronary artery disease and peripheral vascular disease, for example.

“Angiogenesis” broadly refers to the process of developing new bloodvessels. The process involves proliferation, migration and tissueinfiltration of capillary endothelial cells from pre-existing bloodvessels. Angiogenesis is important in normal physiological processes,including for example, follicular growth, embryonal development andwound healing and in pathological processes such as tumor growth andmetastasis. The term “modulation” refers to a change in extent,duration, levels, or properties of a physiologic process. For examplemodulation of angiogenesis could comprise an increase in the formationof new blood vessels or a decrease in the formation of new bloodvessels. Modulation of angiogenesis could also refer to the stimulationof the formation of nonpermeable or nonhyperpermeable blood vessels.Various assays for angiogenesis are described infra.

The terms “treating” and “treatment” as used herein refer to reductionin severity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage.

By an “effective” amount (or “therapeutically effective” amount) of apharmaceutical composition is meant a sufficient, but nontoxic amount ofthe agent to provide the desired effect. The term refers to an amountsufficient to treat a subject. Thus, the term therapeutic amount refersto an amount sufficient to remedy a disease state or symptoms, bypreventing, hindering, retarding or reversing the progression of thedisease or any other undesirable symptoms whatsoever. The termprophylactically effective amount refers to an amount given to a subjectthat does not yet have the disease, and thus is an amount effective toprevent, hinder or retard the onset of a disease.

A “wound” refers broadly to trauma to any tissue. A wound can resultfrom an injury such as caused by violence, accident or surgery. A woundcan include laceration or breakage of a epithelial layer (e.g., theskin) with damage to underlying tissue.

II. Overview

A variety of methods are provided herein for modulating angiogenesis andtreating ischemia. In some instances, such methods involve contacting acell or population of cells such as in an organism, with one or morezinc finger proteins (ZFPs) that bind to specific sequences in one ormore VEGF genes. In certain methods, one ZFP is administered and is ableto bind to a target site in different VEGF genes (e.g., a target site inVEGF-A, VEGF-B and VEGF-C). Other methods involve administering aplurality of different ZFPs that bind to a multiple target sites withina particular gene.

Thus, also provided herein are a variety of zinc finger proteins thatare engineered to specifically recognize and bind to particular nucleicacid segments (target sites), thereby modulating the expression of oneor more VEGF genes. In one embodiment, the ZFPs are linked to regulatorydomains to create chimeric transcription factors to activate or represstranscription of VEGF genes. With such ZFPs, expression of certain VEGFgenes can be enhanced; with certain other ZFPs, expression can berepressed. In general, the target sites to which the ZFPs bind are sitesthat result in activation or repression of expression of VEGF gene. Thetarget site can be adjacent to, upstream of, and/or downstream of thetranscription start site (defined as nucleotide 0). As indicated above,some of the present ZFPs modulate the expression of a single VEGF gene.Other ZFPs modulate the expression of a plurality of VEGF genes. Thus,depending upon the particular ZFP(s) utilized, one can tailor the levelat which one or more VEGF genes are expressed. In addition, multipleZFPs or ZFP fusion molecules, having distinct target sites, can be usedto regulate a single VEGF gene.

By virtue of the ability of the ZFPs to bind to target sites andinfluence expression of VEGF genes, coupled with the diverse functionsof VEGF genes, the ZFPs provided herein can be used in a wide variety ofapplications. In general, the ZFPs can be used to regulate the growth ofa variety of endothelial cells, either by activating or repressinggrowth. In certain applications, the ZFPs can be used to activateexpression of VEGF genes to trigger beneficial angiogenesis in cellpopulations, both in vitro and in vivo. Such activation can be utilizedfor example to promote the formation of new blood vessels andcapillaries in treatments for ischemic conditions. For instance, theZFPs can be used to stimulate the development of collateral circulationin individuals having any of a variety of arterial or venousobstructions, such as individuals having arthrosclerosis. The ZFPs canalso be used to promote lymphogenesis (the formation of lymphaticvessels) and myelopoiesis (the formation of the tissue elements of bonemarrow and/or types of blood cells derived from bone marrow), and can beused to regenerate blood vessels and tissue in wounds.

Other methods involve repression of angiogenesis. Hence, the ability toutilize certain of the ZFPs provided herein to repress the expression ofVEGF genes can be useful in other instances. For example, the ZFPs canbe administered to prevent endothelial growth when such growth isundesirable, such as in blocking the formation of additional bloodvessels to tumors, in preventing proliferation of the microvascularsystem in pathologies such as diabetic retinopathy and pathologicalangiogenesis associated with arthritis.

The ZFPs can also be employed in applications other than therapeuticapplications. For instance, the ZFPs can be used to screen for agentscapable of countering either activation or repression of VEGF geneexpression. Also described herein are nucleic acids that encode the zincfinger proteins. Additionally, agents identified through the screeningmethods, the nucleic acids encoding the ZFPs and/or the ZFPs themselvescan be utilized in pharmaceutical compositions to treat a variety ofdisorders, such as those just described.

III. Zinc Finger Proteins for Regulating Gene Expression

A. General

The zinc finger proteins (ZFPs) disclosed herein are proteins that canbind to DNA in a sequence-specific manner. As indicated supra, theseZFPs can be used in a variety of applications, including modulatingangiogenesis and in treatments for ischemia. An exemplary motifcharacterizing one class of these proteins, the C2H2 class, is-Cys-(X)2-4-Cys-(X)12-His-(X)3-5-His (where X is any amino acid) (SEQ.ID. NO:208). Several structural studies have demonstrated that thefinger domain contains an alpha helix containing the two invarianthistidine residues and two invariant cysteine residues in a beta turncoordinated through zinc. However, the ZFPs provided herein are notlimited to this particular class. Additional classes of zinc fingerproteins are known and can also be used in the methods and compositionsdisclosed herein (see, e.g., Rhodes, et al. (1993) Scientific American268:56-65). In certain ZFPs, a single finger domain is about 30 aminoacids in length. Zinc finger domains are involved not only inDNA-recognition, but also in RNA binding and in protein-protein binding.

The x-ray crystal structure of Zif268, a three-finger domain from amurine transcription factor, has been solved in complex with a cognateDNA-sequence and shows that each finger can be superimposed on the nextby a periodic rotation. The structure suggests that each fingerinteracts independently with DNA over 3 base-pair intervals, withside-chains at positions −1, 2, 3 and 6 on each recognition helix makingcontacts with their respective DNA triplet subsites. The amino terminusof Zif268 is situated at the 3′ end of the DNA strand with which itmakes most contacts. Some zinc fingers can bind to a fourth base in atarget segment. If the strand with which a zinc finger protein makesmost contacts is designated the target strand, some zinc finger proteinsbind to a three base triplet in the target strand and a fourth base onthe nontarget strand. The fourth base is complementary to the baseimmediately 3′ of the three base subsite.

B. Exemplary ZFPs

ZFPs that bind to particular target sites on a nucleic acid thatincludes a VEGF gene are disclosed herein. Thus, the ZFPs can include avariety of different component fingers of varying amino acidcomposition, provided the ZFP binds to the target sites provided herein.Locations of target sites in several VEGF genes are given in Table 2 andthe sequences of the target sites are listed in Tables 3 and 4 below incolumn 2 labeled “Target” in each of these two tables. Transcriptionstart sites for certain of the VEGF genes have not yet been preciselyestablished. Thus, the site location listed in Table 2 for the followingVEGF genes assumes that the transcription start site is at the locationindicted for a particular GenBank entry as follows:

Gene Assumed Transcription Start Site

VEGF A by 2363 of GenBank entry AF095785

VEGF B by 400 of GenBank entry U80601

VEGF-C by 600 of GenBank entry AF020393

VEGF-D by 18727 of GenBank entry U69570

VEGF-E by 30278 (antisense strand) of GenBank entry AC015837

Viral VEGFs by 230 of GenBank entry S67520

As indicated supra, the target sites can be located upstream ordownstream of the transcriptional start site (defined as nucleotide 0).Some of the target sites include 9 nucleotides (see Table 3), whereasother sites include 18 nucleotides (see Table 4). One feature of thesetarget sites is that binding of a ZFP, or a fusion protein including aZFP and one or more regulatory domains, to the target site can affectthe level of expression of one or more VEGF genes. As defined supra,VEGF genes that can be regulated by the ZFPs provided herein include,but are not limited to, VEGF-A (including isoforms VEGF-A121, VEGF-A145,VEGF-A165, VEGF-A189, and VEGF-A206), VEGF B (including isoformsVEGF-B167, and VEGF-B186), VEGF C, VEGF D, the viral VEGF-like proteins(viral VEGF-E) and mammalian VEGF-E, VEGF-H, VEGF-R, VEGF-X, VEGF-138and P1GF (including P1GF-1 and P1GF-2). The target sites can be locatedadjacent the transcription start site or be located significantlyupstream or downstream of the transcription start site. Some targetsites are located within a single VEGF gene such that binding of a ZFPto the target affects the expression of a single VEGF gene. Other targetsites are located within multiple VEGF genes such that the binding of asingle ZFP can modulate the expression of multiple genes. In still otherinstances multiple ZFPs can be used, each recognizing targets in thesame gene.

The ZFPs that bind to these target sites typically include at least onezinc finger but can include a plurality of zinc fingers (e.g., 2, 3, 4,5, 6 or more fingers). Usually, the ZFPs include at least three fingers.Certain of the ZFPs include six fingers. The ZFPs that include threefingers typically recognize a target site that includes 9 or 10nucleotides; ZFPs having six fingers can recognize target sites thatinclude 18 to 20 nucleotides. The ZFPs can also be fusion proteins thatinclude one or more regulatory domains, which domains can betranscriptional activation or repressor domains.

Tables 3 and 4 show the amino acid sequences of a number of differentZFPs and the corresponding target sites to which they bind. Table 3lists ZFPs that bind to target sites that include 9 nucleotides. Thefirst column in this table lists an internal reference name of the ZFP.Column 2 includes the 9 base target site bound by a three-finger zincfinger protein, with the target sites listed in 5′ to 3′ orientation.The corresponding SEQ ID NO. for the target site is listed in column 3.The amino acid sequences of portions of the three zinc finger componentsinvolved in recognition are listed in columns 4, 6 and 8, and theircorresponding SEQ ID NOs. are listed in columns 5, 7 and 9,respectively. The numbering convention for zinc fingers is definedbelow. Column 10 lists the dissociation constants for some of theZFP/target site complexes. Methods for determining such constants aredescribed infra. Excluding cross-strand interactions, each finger bindsto a triplet of bases (a target subsite) within a corresponding targetsequence. The first finger binds to the first triplet starting from the3′ end of a target site, the second finger binds to the second triplet,and the third finger binds the third (i.e., the 5′-most) triplet of thetarget sequence. Thus, for example, the RSDHLAR finger (SEQ ID NO30) ofthe ZFP BVO 13A (first column of Table 3) binds to 5′GGG3′, the DRSNLTRfinger (SEQ ID NO: 36) binds to 5′GAC3′ and the RSDALTQ finger (SEQ IDNO:88) binds to 5′ATG3′.

Table 4 provides information on six-finger ZFPs targeting VEGF genes.Table 4 has a similar format to Table 3, with column 1 indicating theinternal reference name of the ZFP. In contrast to Table 3, however,column 2 of Table 4 includes the 18 base target site recognized by asix-finger protein (here, too, targets are listed in a 5′ to 3′orientation), with the corresponding SEQ ID NO. listed in column 3. Theamino acid sequences of portions of the six zinc finger componentsinvolved in recognition are listed in columns 4, 6, 8, 10, 12 and 14,with associated SEQ ID NOs. being listed in columns 5, 7, 9, 11, 13 and15, respectively. In ZFPs of this type, the first finger binds to thefirst triplet starting from the 3′ end of a target site, the secondfinger binds to the second triplet, the third finger binds the thirdtriplet, the fourth finger binds to the fourth triplet, the fifth fingerbinds to the fifth triplet and the sixth finger binds to the sixth(i.e., the 5′-most) triplet of the target sequence (again excludingcross-strand interactions). Hence, for the ZFP named BVO 10A-9A, thefirst finger QSSDLRR (SEQ ID NO:120) binds 5′GCT3′, the second fingerRSDHLTR (SEQ ID NO:123) binds 5′GGG3′, the third finger DRSALAR (SEQ IDNO:126) binds 5′GTC3′, the fourth finger RSDHLAR (SEQ ID NO: 129) binds5′GGG3′, the fifth finger RSDNLAR (SEQ ID NO:132) binds 5′GAG3′ and thesixth finger RSDALTR (SEQ ID NO:135) binds 5′GTG3′.

Accordingly, some of the target sites for the ZFPs provided herein, aswell as specific examples of such ZFPs are disclosed in Tables 3 and 4.The segments or regions of the VEGF genes that were examined forpotential target sites are indicated in Table 1. The numbers listed inTable 1 refer to the starting and ending base (in kbp) relative to thetranscription start site for various VEGF genes that were examined toidentify target sites. Negative numbers refer to kbp upstream of thetranscription start site and positive numbers refer to kbp downstream ofthe transcription start site, where the transcription start site isdefined as nucleotide 0. The VEGF sequences examined for target sitesinclude the sequences for VEGF-A (see GenBank accession numberAF095785), VEGF-B (see GenBank accession number U80601—from −0.4 kb to+0.32 kb), VEGF-C (see GenBank accession number AF020393) and VEGF-Dgenes (see, HSU69570 and HSY12864), as well as the sequences for P1 GF(see, GenBank accession number AC015837) and viral VEGF-E genes (see,GenBank accession number AF106020 and Meyer, M., et al. (1999) EMBO J.18:363-74; GenBank accession number S67520 and Lyttle, D. J. et al.(1994) J. Virol. 68:84-92; and GenBank accession number AF091434).References providing the sequences for each of these genes are listedsupra. Thus, for example and with reference to Table 1, the nucleotidesequence of the VEGF-A gene examined for target sites extended from 2.3kb upstream of the transcriptional start site to 1.1 kb downstream ofthe transcriptional start site.

The location(s) of the target site(s) for a particular ZFP in thevarious VEGF genes is summarized in Table 2. The first column in thistable is an internal reference name for a ZFP and corresponds to thesame name in column 1 of Tables 3 and 4. The location of the 5′ end ofthe target site in various VEGF gene sequences is listed in theremaining columns. Again, negative numbers refer to the number ofnucleotides upstream of the transcriptional start site (defined asnucleotide 0), whereas positive numbers indicate the number ofnucleotides downstream of the transcriptional start site. Hence, thetarget site (5′ end) for the ZFP named BVO 13A (row 1 of Table 2) in theVEGF A nucleotide sequence begins at the nucleotide located 851 basesdownstream of the principal transcriptional start site for VEGF A.Certain target sites appear in multiple VEGF genes. For instance, thetarget site (5′ end) for the ZFP named VG 4A in the VEGF A nucleotidesequence begins at the nucleotide located 1083 bases upstream of thetranscriptional start site for VEGF A. The same target site (5′ end)also begins at nucleotide 31 upstream of the transcriptional start sitefor VEGF B and 252 bases upstream of the transcription start site forVEGF C. Certain ZFPs, have more than one target site in a single VEGFgene; e.g., EP 10A has two target sites in the VEGF A gene (see Table2).

Consequently, as indicated supra, certain ZFPs described herein can beutilized to modulate angiogenesis by modulating the activity of singleVEGF genes, while other ZFPs can be utilized to regulate expression of aplurality of genes. The ZFP referred to as VG 8A, for example, has abinding site for each of the VEGF genes listed in Table 2, and thus canbe utilized to regulate expression of a variety of VEGF genessimultaneously. By judicious selection of the various ZFPs providedherein and/or combinations thereof, one can tailor which VEGF genes aremodulated.

IV. Characteristics of ZFPs

Zinc finger proteins are formed from zinc finger components. Forexample, zinc finger proteins can have one to thirty-seven fingers,commonly having 2, 3, 4, 5 or 6 fingers. A zinc finger proteinrecognizes and binds to a target site (sometimes referred to as a targetsegment) that represents a relatively small subsequence within a targetgene. Each component finger of a zinc finger protein can bind to asubsite within the target site. The subsite includes a triplet of threecontiguous bases all on the same strand (sometimes referred to as thetarget strand). The subsite may or may not also include a fourth base onthe opposite strand that is the complement of the base immediately 3′ ofthe three contiguous bases on the target strand. In many zinc fingerproteins, a zinc finger binds to its triplet subsite substantiallyindependently of other fingers in the same zinc finger protein.Accordingly, the binding specificity of zinc finger protein containingmultiple fingers is usually approximately the aggregate of thespecificities of its component fingers. For example, if a zinc fingerprotein is formed from first, second and third fingers that individuallybind to triplets XXX, YYY, and ZZZ, the binding specificity of the zincfinger protein is 3′XXX YYY ZZZ5′.

The relative order of fingers in a zinc finger protein from N-terminalto C-terminal determines the relative order of triplets in the 3′ to 5′direction in the target. For example, if a zinc finger protein comprisesfrom N-terminal to C-terminal first, second and third fingers thatindividually bind, respectively, to triplets 5′ GAC3′, 5′GTA3′ and5″GGC3′ then the zinc finger protein binds to the target segment3′CAGATGCGG5′ (SEQ ID NO: 209). If the zinc finger protein comprises thefingers in another order, for example, second finger, first finger,third finger, then the zinc finger protein binds to a target segmentcomprising a different permutation of triplets, in this example,3′ATGCAGCGG5′ (SEQ ID NO: 210). See Berg & Shi, Science 271, 1081-1086(1996). The assessment of binding properties of a zinc finger protein asthe aggregate of its component fingers may, in some cases, be influencedby context-dependent interactions of multiple fingers binding in thesame protein.

Two or more zinc finger proteins can be linked to have a targetspecificity that is the aggregate of that of the component zinc fingerproteins (see e.g., Kim & Pabo, Proc. Natl. Acad. Sci. U.S.A. 95,2812-2817 (1998)). For example, a first zinc finger protein havingfirst, second and third component fingers that respectively bind to XXX,YYY and ZZZ can be linked to a second zinc finger protein having first,second and third component fingers with binding specificities, AAA, BBBand CCC. The binding specificity of the combined first and secondproteins is thus 3′XXXYYYZZZ_AAABBBCCC5′, where the underline indicatesa short intervening region (typically 0-5 bases of any type). In thissituation, the target site can be viewed as comprising two targetsegments separated by an intervening segment.

Linkage can be accomplished using any of the following peptide linkers.

T G E K P: (SEQ ID NO:211) (Liu et al., 1997, supra.); (G4S)_(n) (SEQ IDNO:212) (Kim et al., Proc. Natl. Acad. Sci. U.S.A. 93: 1156-1160 (1996);GGRRGGGS; (SEQ ID NO:213) LRQRDGERP; (SEQ ID NO:214) LRQKDGGGSERP; (SEQID NO:215) LRQKD(G3S)2ERP (SEQ ID NO:216) Alternatively, flexiblelinkers can be rationally designed using computer programs capable ofmodeling both DNA-binding sites and the peptides themselves or by phagedisplay methods. In a further variation, noncovalent linkage can beachieved by fusing two zinc finger proteins with domains promotingheterodimer formation of the two zinc finger proteins. For example, onezinc finger protein can be fused with fos and the other with jun (seeBarbas et al., WO 95/119431).

Linkage of two zinc finger proteins is advantageous for conferring aunique binding specificity within a mammalian genome. A typicalmammalian diploid genome consists of 3×109 bp. Assuming that the fournucleotides A, C, G, and T are randomly distributed, a given 9 bysequence is present ˜23,000 times. Thus a ZFP recognizing a 9 by targetwith absolute specificity would have the potential to bind to ˜23,000sites within the genome. An 18 by sequence is present once in 3.4×1010bp, or about once in a random DNA sequence whose complexity is ten timesthat of a mammalian genome.

A component finger of zinc finger protein typically contains about 30amino acids and, in one embodiment, has the following motif (N-C):

(SEQ ID NO: 208)  Cys-(X)2-4-Cys-X.X.X.X.X.X.X.X.X.X.X.X-His-(X)3- −1 12 3 4 5 6 7 5-His

The two invariant histidine residues and two invariant cysteine residuesin a single beta turn are co-ordinated through zinc atom (see, e.g.,Berg & Shi, Science 271, 1081-1085 (1996)). The above motif shows anumbering convention that is standard in the field for the region of azinc finger conferring binding specificity. The amino acid on the left(N-terminal side) of the first invariant His residue is assigned thenumber +6, and other amino acids further to the left are assignedsuccessively decreasing numbers. The alpha helix begins at residue 1 andextends to the residue following the second conserved histidine. Theentire helix is therefore of variable length, between 11 and 13residues.

V. Design of ZFPs

The ZFPs provided herein are engineered to recognize a selected targetsite in a VEGF gene such as shown in Tables 3, 4 and 6. The process ofdesigning or selecting a ZFP typically starts with a natural ZFP as asource of framework residues. The process of design or selection servesto define nonconserved positions (i.e., positions −1 to +6) so as toconfer a desired binding specificity. One suitable ZFP is the DNAbinding domain of the mouse transcription factor Zif268. The DNA bindingdomain of this protein has the amino acid sequence:

(SEQ ID NO: 217) YACPVESCDRRFSRSDELTRHIRIHTGQKP (F1) (SEQ ID NO: 218)FQCRICMRNFSRSDHLTTHIRTHTGEKP (F2) SEQ ID NO: 219)FACDICGRKFARSDERKRHTKIHLRQK (F3) and (SEQ ID NO: 220) binds to a target5′ GCG TGG GCG 3′.

Another suitable natural zinc finger protein as a source of frameworkresidues is Sp-1. The Sp-1 sequence used for construction of zinc fingerproteins corresponds to amino acids 531 to 624 in the Sp-1 transcriptionfactor. This sequence is 94 amino acids in length. The amino acidsequence of Sp-1 is as follows:

(SEQ ID NO: 221) PGKKKQHICHIQGCGKVYGKTSHLRAHLRWHTGERPFMCTWSYCGKRFTRSDELQRHKRTHTGEKKFACPECPKRFMRSDHLSKHIKTHQNKKG

Sp-1 binds to a target site 5′GGG GCG GGG3′ (SEQ ID No:222).

An alternate form of Sp-1, an Sp-1 consensus sequence, has the followingamino acid sequence:

(SEQ ID NO: 223) meklrngsgdPGKKKQHACPECGKSFSKSSHLRAHQRTHTGERPYKCPECGKSFSRSDELQRHQRTHTGEKPYKCPECGKSFSRSDHLSKHQRTHQNKKG

-   -   (lower case letters are a leader sequence from Shi & Berg,        Chemistry and Biology 1, 83-89. (1995). The optimal binding        sequence for the Sp-1 consensus sequence is 5′ GGGGCGGGG3′ (SEQ        ID NO:222). Other suitable ZFPs are described below.

There are a number of substitution rules that assist rational design ofsome zinc finger proteins. For example, ZFP DNA-binding domains can bedesigned and/or selected to recognize a particular target site asdescribed in co-owned WO 00/42219; WO 00/41566; and U.S. Ser. No.09/444,241 filed Nov. 19, 1999; 09/535,088 filed Mar. 23, 2000; as wellas U.S. Pat. Nos. 5,789,538; 6,007,408; 6,013,453; 6,140,081; and6,140,466; and PCT publications WO 95/19431, WO 98/54311, WO 00/23464and WO 00/27878. In one embodiment, a target site for a zinc fingerDNA-binding domain is identified according to site selection rulesdisclosed in co-owned WO 00/42219. In a preferred embodiment, a ZFP isselected as described in co-owned U.S. Ser. No. 09/716,637, filed Nov.20, 2000, titled “Iterative Optimization in the Design of BindingProteins.” See also WO 96/06166; Desjarlais & Berg, PNAS 90, 2256-2260(1993); Choo & Klug, PNAS 91, 11163-11167 (1994); Desjarlais & Berg,PNAS 89, 7345-7349 (1992); Jamieson et al., Biochemistry 33:5689-5695(1994); and Choo et al., WO 98/53057, WO 98/53058; WO 98/53059; WO98/53060.

Many of these rules are supported by site-directed mutagenesis of thethree-finger domain of the ubiquitous transcription factor, Sp-1(Desjarlais and Berg, 1992; 1993). One of these rules is that a 5′ G ina DNA triplet can be bound by a zinc finger incorporating arginine atposition 6 of the recognition helix. Another substitution rule is that aG in the middle of a subsite can be recognized by including a histidineresidue at position 3 of a zinc finger. A further substitution rule isthat asparagine can be incorporated to recognize A in the middle of atriplet, aspartic acid, glutamic acid, serine or threonine can beincorporated to recognize C in the middle of a triplet, and amino acidswith small side chains such as alanine can be incorporated to recognizeT in the middle of a triplet. A further substitution rule is that the 3′base of a triplet subsite can be recognized by incorporating thefollowing amino acids at position −1 of the recognition helix: arginineto recognize G, glutamine to recognize A, glutamic acid (or asparticacid) to recognize C, and threonine to recognize T. Although thesesubstitution rules are useful in designing zinc finger proteins they donot take into account all possible target sites. Furthermore, theassumption underlying the rules, namely that a particular amino acid ina zinc finger is responsible for binding to a particular base in asubsite is only approximate. Context-dependent interactions betweenproximate amino acids in a finger or binding of multiple amino acids toa single base or vice versa can cause variation of the bindingspecificities predicted by the existing substitution rules. Accordingly,in certain embodiments, a ZFP DNA-binding domain of predeterminedspecificity is obtained according to the methods described in co-ownedU.S. Ser. No. 09/716,637, filed Nov. 20, 2000, titled “IterativeOptimization in the Design of Binding Proteins.”

Any suitable method known in the art can be used to design and constructnucleic acids encoding ZFPs, e.g., phage display, random mutagenesis,combinatorial libraries, computer/rational design, affinity selection,PCR, cloning from cDNA or genomic libraries, synthetic construction andthe like. (see, e.g., U.S. Pat. No. 5,786,538; Wu et al., PNAS92:344-348 (1995); Jamieson et al., Biochemistry 33:5689-5695 (1994);Rebar & Pabo, Science 263:671-673 (1994); Choo & Klug, PNAS91:11163-11167 (1994); Choo & Klug, PNAS 91: 11168-11172 (1994);Desjarlais & Berg, PNAS 90:2256-2260 (1993); Desjarlais & Berg, PNAS89:7345-7349 (1992); Pomerantz et al., Science 267:93-96 (1995);Pomerantz et al., PNAS 92:9752-9756 (1995); and Liu et al., PNAS94:5525-5530 (1997); Griesman & Pabo, Science 275:657-661 (1997);Desjarlais & Berg, PNAS 91:11-99-11103 (1994)).

In certain preferred embodiments, the binding specificity of aDNA-binding domain (e.g., a ZFP DNA-binding domain) is determined byidentifying accessible regions in the sequence in question (e.g., incellular chromatin). Accessible regions can be determined as describedin co-owned U.S. Patent Application Ser. No. 60/228,556 entitled“Databases of Accessible Region Sequences; Methods of Preparation andUse Thereof,” filed Aug. 28, 2000, the disclosure of which is herebyincorporated by reference herein. See also Example 1. A DNA-bindingdomain is then designed and/or selected as described herein to bind to atarget site within the accessible region

VI. Production of Zinc Finger Proteins

A. Synthesis and Cloning

ZFP polypeptides and nucleic acids encoding the same can be made usingroutine techniques in the field of recombinant genetics. Basic textsdisclosing general methods include Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)). In addition, nucleicacids less than about 100 bases can be custom ordered from any of avariety of commercial sources, such as The Midland Certified ReagentCompany (mcrc@oligos.com), The Great American Gene Company(http://www.genco.com), ExpressGen Inc. (www.expressgen.com), OperonTechnologies Inc. (Alameda, Calif.). Similarly, peptides can be customordered from any of a variety of sources, such as PeptidoGenic(pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMABiomedicals Ltd (U.K.), Bio.Synthesis, Inc.

Oligonucleotides can be chemically synthesized according to the solidphase phosphoramidite triester method first described by Beaucage &Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Purification of oligonucleotides is by eitherdenaturing polyacrylamide gel electrophoresis or by reverse phase HPLC.The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

Two alternative methods are typically used to create the codingsequences required to express newly designed DNA-binding peptides. Oneprotocol is a PCR-based assembly procedure that utilizes six overlappingoligonucleotides. Three oligonucleotides correspond to “universal”sequences that encode portions of the DNA-binding domain between therecognition helices. These oligonucleotides typically remain constantfor all zinc finger constructs. The other three “specific”oligonucleotides are designed to encode the recognition helices. Theseoligonucleotides contain substitutions primarily at positions −1, 2, 3and 6 on the recognition helices making them specific for each of thedifferent DNA-binding domains.

The PCR synthesis is carried out in two steps. First, a double strandedDNA template is created by combining the six oligonucleotides (threeuniversal, three specific) in a four cycle PCR reaction with a lowtemperature annealing step, thereby annealing the oligonucleotides toform a DNA “scaffold.” The gaps in the scaffold are filled in byhigh-fidelity thermostable polymerase, the combination of Taq and Pfupolymerases also suffices. In the second phase of construction, the zincfinger template is amplified by external primers designed to incorporaterestriction sites at either end for cloning into a shuttle vector ordirectly into an expression vector.

An alternative method of cloning the newly designed DNA-binding proteinsrelies on annealing complementary oligonucleotides encoding the specificregions of the desired ZFP. This particular application requires thatthe oligonucleotides be phosphorylated prior to the final ligation step.This is usually performed before setting up the annealing reactions. Inbrief, the “universal” oligonucleotides encoding the constant regions ofthe proteins (oligos 1, 2 and 3 of above) are annealed with theircomplementary oligonucleotides. Additionally, the “specific”oligonucleotides encoding the finger recognition helices are annealedwith their respective complementary oligonucleotides. Thesecomplementary oligos are designed to fill in the region which waspreviously filled in by polymerase in the above-mentioned protocol.Oligonucleotides complementary to oligos 1 and 6 are engineered to leaveoverhanging sequences specific for the restriction sites used in cloninginto the vector of choice in the following step. The second assemblyprotocol differs from the initial protocol in the following aspects: the“scaffold” encoding the newly designed ZFP is composed entirely ofsynthetic DNA thereby eliminating the polymerase fill-in step,additionally the fragment to be cloned into the vector does not requireamplification. Lastly, the design of leaving sequence-specific overhangseliminates the need for restriction enzyme digests of the insertingfragment. Alternatively, changes to ZFP recognition helices can becreated using conventional site-directed mutagenesis methods.

Both assembly methods require that the resulting fragment encoding thenewly designed ZFP be ligated into a vector. Ultimately, theZFP-encoding sequence is cloned into an expression vector. Expressionvectors that are commonly utilized include, but are not limited to, amodified pMAL-c2 bacterial expression vector (New England BioLabs,Beverly, Mass.) or an eukaryotic expression vector, pcDNA (Promega,Madison, Wis.). The final constructs are verified by sequence analysis.

Any suitable method of protein purification known to those of skill inthe art can be used to purify ZFPs (see, Ausubel, supra, Sambrook,supra). In addition, any suitable host can be used for expression, e.g.,bacterial cells, insect cells, yeast cells, mammalian cells, and thelike.

Expression of a zinc finger protein fused to a maltose binding protein(MBP-ZFP) in bacterial strain JM109 allows for straightforwardpurification through an amylose column (New England BioLabs, Beverly,Mass.). High expression levels of the zinc finger chimeric protein canbe obtained by induction with IPTG since the MBP-ZFP fusion in thepMal-c2 expression plasmid is under the control of the tac promoter (NewEngland BioLabs, Beverly, Mass.). Bacteria containing the MBP-ZFP fusionplasmids are inoculated into 2xYT medium containing 10 μM ZnCl2, 0.02%glucose, plus 50 μg/ml ampicillin and shaken at 37° C. Atmid-exponential growth IPTG is added to 0.3 mM and the cultures areallowed to shake. After 3 hours the bacteria are harvested bycentrifugation, disrupted by sonication or by passage through a frenchpressure cell or through the use of lysozyme, and insoluble material isremoved by centrifugation. The MBP-ZFP proteins are captured on anamylose-bound resin, washed extensively with buffer containing 20 mMTris-HCl (pH 7.5), 200 mM NaCl, 5 mM DTT and 50 μM ZnCl2, then elutedwith maltose in essentially the same buffer (purification is based on astandard protocol from New England BioLabs. Purified proteins arequantitated and stored for biochemical analysis.

The dissociation constant of a purified protein, e.g., Kd, is typicallycharacterized via electrophoretic mobility shift assays (EMSA)(Buratowski & Chodosh, in Current Protocols in Molecular Biology pp.12.2.1-12.2.7 (Ausubel ed., 1996)). Affinity is measured by titratingpurified protein against a fixed amount of labeled double-strandedoligonucleotide target. The target typically comprises the naturalbinding site sequence flanked by the 3 by found in the natural sequenceand additional, constant flanking sequences. The natural binding site istypically 9 by for a three-finger protein and 2×9 by+intervening basesfor a six finger ZFP. The annealed oligonucleotide targets possess a 1base 5′ overhang which allows for efficient labeling of the target withT4 phage polynucleotide kinase. For the assay the target is added at aconcentration of 1 nM or lower (the actual concentration is kept atleast 10-fold lower than the expected dissociation constant), purifiedZFPs are added at various concentrations, and the reaction is allowed toequilibrate for at least 45 min. In addition the reaction mixture alsocontains 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM MgCl2, 0.1 mM ZnCl2, 5 mMDTT, 10% glycerol, 0.02% BSA.

The equilibrated reactions are loaded onto a 10% polyacrylamide gel,which has been pre-run for 45 min in Tris/glycine buffer, then bound andunbound labeled target is resolved by electrophoresis at 150V.Alternatively, 10-20% gradient Tris-HCl gels, containing a 4%polyacrylamide stacking gel, can be used. The dried gels are visualizedby autoradiography or phosphorimaging and the apparent Kd is determinedby calculating the protein concentration that yields half-maximalbinding.

The assays can also include a determination of the active fraction inthe protein preparations. Active fraction is determined bystoichiometric gel shifts in which protein is titrated against a highconcentration of target DNA. Titrations are done at 100, 50, and 25% oftarget (usually at micromolar levels).

B. Phage Display

The technique of phage display provides a largely empirical means ofgenerating zinc finger proteins with a desired target specificity (seee.g., Rebar, U.S. Pat. No. 5,789,538; Choo et al., WO 96/06166; Barbaset al., WO 95/19431 and WO 98/543111; Jamieson et al., supra). Themethod can be used in conjunction with, or as an alternative to rationaldesign. The method involves the generation of diverse libraries ofmutagenized zinc finger proteins, followed by the isolation of proteinswith desired DNA-binding properties using affinity selection methods. Touse this method, the experimenter typically proceeds as follows. First,a gene for a zinc finger protein is mutagenized to introduce diversityinto regions important for binding specificity and/or affinity. In atypical application, this is accomplished via randomization of a singlefinger at positions −1, +2, +3, and +6, and sometimes accessorypositions such as +1, +5, +8 and +10. Next, the mutagenized gene iscloned into a phage or phagemid vector as a fusion with gene III of afilamentous phage, which encodes the coat protein pIII. The zinc fingergene is inserted between segments of gene III encoding the membraneexport signal peptide and the remainder of pIII, so that the zinc fingerprotein is expressed as an amino-terminal fusion with pill or in themature, processed protein. When using phagemid vectors, the mutagenizedzinc finger gene may also be fused to a truncated version of gene IIIencoding, minimally, the C-terminal region required for assembly of pIIIinto the phage particle. The resultant vector library is transformedinto E. coli and used to produce filamentous phage which express variantzinc finger proteins on their surface as fusions with the coat proteinpIII. If a phagemid vector is used, then the this step requiressuperinfection with helper phage. The phage library is then incubatedwith a target DNA site, and affinity selection methods are used toisolate phage which bind target with high affinity from bulk phage.Typically, the DNA target is immobilized on a solid support, which isthen washed under conditions sufficient to remove all but the tightestbinding phage. After washing, any phage remaining on the support arerecovered via elution under conditions which disrupt zinc finger—DNAbinding. Recovered phage are used to infect fresh E. coli., which isthen amplified and used to produce a new batch of phage particles.Selection and amplification are then repeated as many times as isnecessary to enrich the phage pool for tight binders such that these maybe identified using sequencing and/or screening methods. Although themethod is illustrated for pIII fusions, analogous principles can be usedto screen ZFP variants as pVIII fusions.

In certain embodiments, the sequence bound by a particular zinc fingerprotein is determined by conducting binding reactions (see, e.g.,conditions for determination of Kd, supra) between the protein and apool of randomized double-stranded oligonucleotide sequences. Thebinding reaction is analyzed by an electrophoretic mobility shift assay(EMSA), in which protein-DNA complexes undergo retarded migration in agel and can be separated from unbound nucleic acid. Oligonucleotideswhich have bound the finger are purified from the gel and amplified, forexample, by a polymerase chain reaction. The selection (i.e. bindingreaction and EMSA analysis) is then repeated as many times as desired,with the selected oligonucleotide sequences. In this way, the bindingspecificity of a zinc finger protein having a particular amino acidsequence is determined.

C. Regulatory Domains

Zinc finger proteins are often expressed with an exogenous domain (orfunctional fragment thereof) as fusion proteins. Common domains foraddition to the ZFP include, e.g., transcription factor domains(activators, repressors, co-activators, co-repressors), silencers,oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mosfamily members etc.); DNA repair enzymes and their associated factorsand modifiers; DNA rearrangement enzymes and their associated factorsand modifiers; chromatin associated proteins and their modifiers (e.g.kinases, acetylases and deacetylases); and DNA modifying enzymes (e.g.,methyltransferases, topoisomerases, helicases, ligases, kinases,phosphatases, polymerases, endonucleases) and their associated factorsand modifiers. A preferred domain for fusing with a ZFP when the ZFP isto be used for repressing expression of a target gene is a KRABrepression domain from the human KOX-1 protein (Thiesen et al., NewBiologist 2, 363-374 (1990); Margolin et al., Proc. Natl. Acad. Sci. USA91, 4509-4513 (1994); Pengue et al., Nucl. Acids Res. 22:2908-2914(1994); Witzgall et al., Proc. Natl. Acad. Sci. USA 91, 4514-4518(1994). Preferred domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Seifpalet al., EMBO J. 11, 4961-4968 (1992)).

The identification of novel sequences and accessible regions (e.g.,DNase I hypersensitive sites) in VEGF genes allows for the design offusion molecules which facilitate modulation of angiogenesis. Thus, incertain embodiments, the compositions and methods disclosed hereininvolve fusions between a DNA-binding domain specifically targeted toone or more regulatory regions of a VEGF gene and a functional (e.g.,repression or activation) domain (or a polynucleotide encoding such afusion). In this way, the repression or activation domain is broughtinto proximity with a sequence in the VEGF gene that is bound by theDNA-binding domain. The transcriptional regulatory function of thefunctional domain is then able to act on VEGF regulatory sequences.

In additional embodiments, targeted remodeling of chromatin, asdisclosed in co-owned U.S. patent application entitled “TargetedModification of Chromatin Structure,” can be used to generate one ormore sites in cellular chromatin that are accessible to the binding of aDNA binding molecule.

Fusion molecules are constructed by methods of cloning and biochemicalconjugation that are well-known to those of skill in the art. Fusionmolecules comprise a DNA-binding domain and a functional domain (e.g., atranscriptional activation or repression domain). Fusion molecules alsooptionally comprise nuclear localization signals (such as, for example,that from the SV40 medium T-antigen) and epitope tags (such as, forexample, FLAG and hemagglutinin). Fusion proteins (and nucleic acidsencoding them) are designed such that the translational reading frame ispreserved among the components of the fusion.

Fusions between a polypeptide component of a functional domain (or afunctional fragment thereof) on the one hand, and a non-proteinDNA-binding domain (e.g., antibiotic, intercalator, minor groove binder,nucleic acid) on the other, are constructed by methods of biochemicalconjugation known to those of skill in the art. See, for example, thePierce Chemical Company (Rockford, Ill.) Catalogue. Methods andcompositions for making fusions between a minor groove binder and apolypeptide have been described. Mapp et al. (2000) Proc. Natl. Acad.Sci. USA 97:3930-3935.

The fusion molecules disclosed herein comprise a DNA-binding domainwhich binds to a target site in a VEGF gene. In certain embodiments, thetarget site is present in an accessible region of cellular chromatin.Accessible regions can be determined as described, for example, inco-owned U.S. Patent Application Ser. No. 60/228,556. If the target siteis not present in an accessible region of cellular chromatin, one ormore accessible regions can be generated as described in co-owned U.S.patent application entitled “Targeted Modification of ChromatinStructure.” In additional embodiments, the DNA-binding domain of afusion molecule is capable of binding to cellular chromatin regardlessof whether its target site is in an accessible region or not. Forexample, such DNA-binding domains are capable of binding to linker DNAand/or nucleosomal DNA. Examples of this type of “pioneer” DNA bindingdomain are found in certain steroid receptor and in hepatocyte nuclearfactor 3 (HNF3). Cordingley et al. (1987) Cell 48:261-270; Pina et al.(1990) Cell 60:719-731; and Cirillo et al. (1998) EMBO J. 17:244-254.

For such applications, the fusion molecule is typically formulated witha pharmaceutically acceptable carrier, as is known to those of skill inthe art. See, for example, Remington's Pharmaceutical Sciences, 17thed., 1985; and co-owned WO 00/42219.

The functional component/domain of a fusion molecule can be selectedfrom any of a variety of different components capable of influencingtranscription of a gene once the fusion molecule binds to a targetsequence via its DNA binding domain. Hence, the functional component caninclude, but is not limited to, various transcription factor domains,such as activators, repressors, co-activators, co-repressors, andsilencers.

An exemplary functional domain for fusing with a DNA-binding domain suchas, for example, a ZFP, to be used for repressing expression of a geneis a KRAB repression domain from the human KOX-1 protein (see, e.g.,Thiesen et al., New Biologist 2, 363-374 (1990); Margolin et al., Proc.Natl. Acad. Sci. USA 91, 4509-4513 (1994); Pengue et al., Nucl. AcidsRes. 22:2908-2914 (1994); Witzgall et al., Proc. Natl. Acad. Sci. USA91, 4514-4518 (1994). Another suitable repression domain is methylbinding domain protein 2B (MBD-2B) (see, also Hendrich et al. (1999)Mamm Genome 10:906-912 for description of MBD proteins). Another usefulrepression domain is that associated with the v-ErbA protein. See, forexample, Damm, et al. (1989) Nature 339:593-597; Evans (1989) Int. J.Cancer Suppl. 4:26-28; Pain et al. (1990) New Biol. 2:284-294; Sap etal. (1989) Nature 340:242-244; Zenke et al. (1988) Cell 52:107-119; andZenke et al. (1990) Cell 61:1035-1049.

Suitable domains for achieving activation include the HSV VP16activation domain (see, e.g., Hagmann et al., J. Virol. 71, 5952-5962(1997)) nuclear hormone receptors (see, e.g., Torchia et al., Curr.Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of nuclear factorkappa B (Bitko & Barik, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,Neuroreport 8:2937-2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28(1998)), or artificial chimeric functional domains such as VP64 (Seifpalet al., EMBO J. 11, 4961-4968 (1992)).

Additional exemplary activation domains include, but are not limited to,VP16, VP64, p300, CBP, PCAF, SRC1 PvALF, AtHD2A and ERF-2. See, forexample, Robyr et al. (2000) Mol. Endocrinol. 14:329-347; Collingwood etal. (1999) J. Mol. Endocrinol. 23:255-275; Leo et al. (2000) Gene245:1-11; Manteuffel-Cymborowska (1999) Acta Biochim. Pol. 46:77-89;McKenna et al. (1999) J. Steroid Biochem. Mol. Biol. 69:3-12; Malik etal. (2000) Trends Biochem. Sci. 25:277-283; and Lemon et al. (1999)Curr. Opin. Genet. Dev. 9:499-504.

Additional exemplary activation domains include, but are not limited to,OsGAI, HALF-1, C1, AP1, ARF-5, -6, -7, and -8, CPRF1, CPRF4, MYC-RP/GP,and TRAB1. See, for example, Ogawa et al. (2000) Gene 245:21-29; Okanamiet al. (1996) Genes Cells 1:87-99; Goff et al. (1991) Genes Dev.5:298-309; Cho et al. (1999) Plant Mol. Biol. 40:419-429; Ulmason et al.(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al.(2000) Plant J. 22:1-8; Gong et al. (1999) Plant Mol. Biol. 41:33-44;and Hobo et al. (1999) Proc. Natl. Acad. Sci. USA 96:15,348-15,353.

Additional exemplary repression domains include, but are not limited to,KRAB, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A,DNMT3B), Rb, and MeCP2. See, for example, Bird et al. (1999) Cell99:451-454; Tyler et al. (1999) Cell 99:443-446; Knoepfler et al. (1999)Cell 99:447-450; and Robertson et al. (2000) Nature Genet. 25:338-342.Additional exemplary repression domains include, but are not limited to,ROM2 and AtHD2A. See, for example, Chem et al. (1996) Plant Cell8:305-321; and Wu et al. (2000) Plant J. 22:19-27.

Additional functional domains are disclosed, for example, in co-owned WO00/41566.

D. Expression Vectors

The nucleic acid encoding the ZFP of choice is typically cloned intointermediate vectors for transformation into prokaryotic or eukaryoticcells for replication and/or expression, e.g., for determination of Kd.Intermediate vectors are typically prokaryote vectors, e.g., plasmids,or shuttle vectors, or insect vectors, for storage or manipulation ofthe nucleic acid encoding ZFP or production of protein. The nucleic acidencoding a ZFP is also typically cloned into an expression vector, foradministration to a plant cell, animal cell, preferably a mammalian cellor a human cell, fungal cell, bacterial cell, or protozoal cell.

To obtain expression of a cloned gene or nucleic acid, a ZFP istypically subcloned into an expression vector that contains a promoterto direct transcription. Suitable bacterial and eukaryotic promoters arewell known in the art and described, e.g., in Sambrook et al., MolecularCloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994). Bacterial expressionsystems for expressing the ZFP are available in, e.g., E. coli, Bacillussp., and Salmonella (Palva et al., Gene 22:229-235 (1983)). Kits forsuch expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

The promoter used to direct expression of a ZFP nucleic acid depends onthe particular application. For example, a strong constitutive promoteris typically used for expression and purification of ZFP. In contrast,when a ZFP is administered in vivo for gene regulation, either aconstitutive or an inducible promoter is used, depending on theparticular use of the ZFP. In addition, a preferred promoter foradministration of a ZFP can be a weak promoter, such as HSV TK or apromoter having similar activity. The promoter typically can alsoinclude elements that are responsive to transactivation, e.g., hypoxiaresponse elements, Gal4 response elements, lac repressor responseelement, and small molecule control systems such as tet-regulatedsystems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547(1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., GeneTher. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); andRendahl et al., Nat. Biotechnol. 16:757-761 (1998)).

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to the nucleicacid sequence encoding the ZFP, and signals required, e.g., forefficient polyadenylation of the transcript, transcriptionaltermination, ribosome binding sites, or translation termination.Additional elements of the cassette may include, e.g., enhancers, andexogenous spliced intronic signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe ZFP. Standard bacterial expression vectors include plasmids such aspBR322 based plasmids, pSKF, pET23D, and commercially available fusionexpression systems such as GST and LacZ. A preferred fusion protein isthe maltose binding protein, “MBP.” Such fusion proteins are used forpurification of the ZFP. Epitope tags can also be added to recombinantproteins to provide convenient methods of isolation, for monitoringexpression, and for monitoring cellular and subcellular localization,e.g., c-myc or FLAG.

Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMT010/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, or otherpromoters shown effective for expression in eukaryotic cells.

Some expression systems have markers for selection of stably transfectedcell lines such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. High yield expression systems are alsosuitable, such as using a baculovirus vector in insect cells, with a ZFPencoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, naked DNA, plasmid vectors, viral vectors,both episomal and integrative, and any of the other well known methodsfor introducing cloned genomic DNA, cDNA, synthetic DNA or other foreigngenetic material into a host cell (see, e.g., Sambrook et al., supra).It is only necessary that the particular genetic engineering procedureused be capable of successfully introducing at least one gene into thehost cell capable of expressing the protein of choice.

VII. Assays

Once a ZFP has been designed and prepared according to the proceduresjust set forth, an initial assessment of the activity of the designedZFP is undertaken. ZFP proteins showing the ability to modulate theexpression of a gene of interest can then be further assayed for morespecific activities depending upon the particular application for whichthe ZFPs have been designed. Thus, for example, the ZFPs provided hereincan be initially assayed for their ability to modulate VEGF expression.More specific assays of the ability of the ZFP to modulate angiogenesisand/or to treat ischemia are then typically undertaken. A description ofthese more specific assays are set forth infra in section IX.

The activity of a particular ZFP can be assessed using a variety of invitro and in vivo assays, by measuring, e.g., protein or mRNA levels,product levels, enzyme activity, tumor growth; transcriptionalactivation or repression of a reporter gene; second messenger levels(e.g., cGMP, cAMP, IP3, DAG, Ca2+); cytokine and hormone productionlevels; and neovascularization, using, e.g., immunoassays (e.g., ELISAand immunohistochemical assays with antibodies), hybridization assays(e.g., RNase protection, Northers, in situ hybridization,oligonucleotide array studies), colorimetric assays, amplificationassays, enzyme activity assays, tumor growth assays, phenotypic assays,and the like.

ZFPs are typically first tested for activity in vitro using culturedcells, e.g., 293 cells, CHO cells, VERO cells, BHK cells, HeLa cells,COS cells, and the like. Preferably, human cells are used. The ZFP isoften first tested using a transient expression system with a reportergene, and then regulation of the target endogenous gene is tested incells and in animals, both in vivo and ex vivo. The ZFP can berecombinantly expressed in a cell, recombinantly expressed in cellstransplanted into an animal, or recombinantly expressed in a transgenicanimal, as well as administered as a protein to an animal or cell usingdelivery vehicles described below. The cells can be immobilized, be insolution, be injected into an animal, or be naturally occurring in atransgenic or non-transgenic animal.

Modulation of gene expression is tested using one of the in vitro or invivo assays described herein. Samples or assays are treated with a ZFPand compared to untreated control samples, to examine the extent ofmodulation. As described above, for regulation of endogenous geneexpression, the ZFP typically has a Kd of 200 nM or less, morepreferably 100 nM or less, more preferably 50 nM, most preferably 25 nMor less.

The effects of the ZFPs can be measured by examining any of theparameters described above. Any suitable gene expression, phenotypic, orphysiological change can be used to assess the influence of a ZFP. Whenthe functional consequences are determined using intact cells oranimals, one can also measure a variety of effects such as tumor growth,wound healing, neovascularization, hormone release, transcriptionalchanges to both known and uncharacterized genetic markers (e.g.,Northern blots or oligonucleotide array studies), changes in cellmetabolism such as cell growth or pH changes, and changes inintracellular second messengers such as cGMP.

Preferred assays for ZFP regulation of endogenous gene expression can beperformed in vitro. In one preferred in vitro assay format, ZFPregulation of endogenous gene expression in cultured cells is measuredby examining protein production using an ELISA assay. The test sample iscompared to control cells treated with a vector lacking ZFP-encodingsequences or a vector encoding an unrelated ZFP that is targeted toanother gene.

In another embodiment, ZFP regulation of endogenous gene expression isdetermined in vitro by measuring the level of target gene mRNAexpression. The level of gene expression is measured usingamplification, e.g., using PCR, LCR, or hybridization assays, e.g.,Northern hybridization, dot blotting and RNase protection. The use ofquantitative RT-PCR techniques (i.e., the so-called TaqMan assays) canalso be utilized to quantitate the level of transcript. The level ofprotein or mRNA is detected using directly or indirectly labeleddetection agents, e.g., fluorescently or radioactively labeled nucleicacids, radioactively or enzymatically labeled antibodies, and the like,as described herein. Such methods are also described in U.S. Pat. Nos.5,210,015 to Gelfand, 5,538,848 to Livak, et al., and 5,863,736 toHaaland, as well as Heid, C. A., et al., Genome Research, 6:986-994(1996); Gibson, U.E.M, et al., Genome Research 6:995-1001 (1996);Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991);and Livak, K. J., et al., PCR Methods and Applications 357-362 (1995),each of which is incorporated by reference in its entirety.

Alternatively, a reporter gene system can be devised using a VEGF genepromoter operably linked to a reporter gene such as luciferase, greenfluorescent protein, CAT, or β-gal. The reporter construct is typicallyco-transfected into a cultured cell. After treatment with the ZFP ofchoice, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art.

Another example of a preferred assay format useful for monitoring ZFPregulation of endogenous gene expression is performed in vivo. Thisassay is particularly useful for examining genes such as VEGF involvedin tumor support via neovascularization. In this assay, cultured tumorcells expressing the ZFP of choice are injected subcutaneously into animmune compromised mouse such as an athymic mouse, an irradiated mouse,or a SCID mouse. After a suitable length of time, preferably 4-8 weeks,tumor growth is measured, e.g., by volume or by its two largestdimensions, and compared to the control. Tumors that have statisticallysignificant reduction (using, e.g., Student's T test) are said to haveinhibited growth. Alternatively, the extent of tumor neovascularizationcan also be measured.

Immunoassays using endothelial cell specific antibodies are used tostain for vascularization of the tumor and the number of vessels in thetumor. Tumors that have a statistically significant reduction in thenumber of vessels (using, e.g., Student's T test) are said to haveinhibited neovascularization.

Transgenic and non-transgenic animals are also used for examiningregulation of VEGF gene expression in vivo. Transgenic animals typicallyexpress the ZFP of choice. Alternatively, animals that transientlyexpress the ZFP of choice, or to which the ZFP has been administered ina delivery vehicle, can be used. Regulation of endogenous geneexpression is tested using any one of the assays described herein.

VIII. Pharmaceutical Compositions

The ZFPs provided herein, and more typically the nucleic acids encodingthem, can optionally be formulated with a pharmaceutically acceptablecarrier as a pharmaceutical composition.

A. Nucleic Acid Based Compositions

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding the present ZFPs in mammalian cellsor target tissues. Such methods can be used to administer nucleic acidsencoding ZFPs to cells in vitro. In some instances, the nucleic acidsencoding ZFPs are administered for in vivo or ex vivo gene therapy uses.Non-viral vector delivery systems include DNA plasmids, naked nucleicacid, and nucleic acid complexed with a delivery vehicle such as aliposome. Viral vector delivery systems include DNA and RNA viruses,which have either episomal or integrated genomes after delivery to thecell. For a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993);Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiologyand Immunology Doerfler and Bohm (eds) (1995); and Yu et al., GeneTherapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids encoding the ZFPsprovided herein include lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S.Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355) and lipofectionreagents are sold commercially (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids that are suitable for efficientreceptor-recognition lipofection of polynucleotides include those ofFeigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivoadministration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFP take advantage of highly evolved processesfor targeting a virus to specific cells in the body and trafficking theviral payload to the nucleus. Viral vectors can be administered directlyto patients (in vivo) or they can be used to treat cells in vitro andthe modified cells are administered to patients (ex vivo). Conventionalviral based systems for the delivery of ZFPs can include retroviral,lentivirus, adenoviral, adeno-associated and herpes simplex virusvectors for gene transfer. Viral vectors are currently the mostefficient and versatile method of gene transfer in target cells andtissues. Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system can thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SW), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the ZFP is preferred,adenoviral based systems are typically used. Adenoviral based vectorsare capable of very high transduction efficiency in many cell types anddo not require cell division. With such vectors, high titer and levelsof expression have been obtained. This vector can be produced in largequantities in a relatively simple system. Adeno-associated virus (“AAV”)vectors are also used to transduce cells with target nucleic acids,e.g., in the in vitro production of nucleic acids and peptides, and forin vivo and ex vivo gene therapy procedures (see, e.g., West et al.,Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin,Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351(1994). Construction of recombinant AAV vectors are described in anumber of publications, including U.S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell.Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984);and Samulski et al., J. Virol. 63:03822-3828 (1989).

In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) is another alternativegene delivery systems based on the defective and nonpathogenicparvovirus adeno-associated type 2 virus. All vectors are derived from aplasmid that retains only the AAV 145 by inverted terminal repeatsflanking the transgene expression cassette. Efficient gene transfer andstable transgene delivery due to integration into the genomes of thetransduced cell are key features for this vector system. (Wagner et al.,Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55(1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used for colon cancer gene therapy, because they can beproduced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., PNAS 92:9747-9751 (1995), reportedthat Moloney murine leukemia virus can be modified to express humanheregulin fused to gp70, and the recombinant virus infects certain humanbreast cancer cells expressing human epidermal growth factor receptor.This principle can be extended to other pairs of virus expressing aligand fusion protein and target cell expressing a receptor. Forexample, filamentous phage can be engineered to display antibodyfragments (e.g., FAB or Fv) having specific binding affinity forvirtually any chosen cellular receptor. Although the above descriptionapplies primarily to viral vectors, the same principles can be appliedto nonviral vectors. Such vectors can be engineered to contain specificuptake sequences thought to favor uptake by specific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In some instances, cells areisolated from the subject organism, transfected with a ZFP nucleic acid(gene or cDNA), and re-infused back into the subject organism (e.g.,patient). Various cell types suitable for ex vivo transfection are wellknown to those of skill in the art (see, e.g., Freshney et al., Cultureof Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and thereferences cited therein for a discussion of how to isolate and culturecells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Tad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions, asdescribed below (see, e.g., Remington's Pharmaceutical Sciences, 17thed., 1989).

B. Protein Compositions

An important factor in the administration of polypeptide compounds, suchas the present ZFPs, is ensuring that the polypeptide has the ability totraverse the plasma membrane of a cell, or the membrane of anintra-cellular compartment such as the nucleus. Cellular membranes arecomposed of lipid-protein bilayers that are freely permeable to small,nonionic lipophilic compounds and are inherently impermeable to polarcompounds, macromolecules, and therapeutic or diagnostic agents.However, proteins and other compounds such as liposomes have beendescribed, which have the ability to translocate polypeptides such asZFPs across a cell membrane.

For example, “membrane translocation polypeptides” have amphiphilic orhydrophobic amino acid subsequences that have the ability to act asmembrane-translocating carriers. In one embodiment, homeodomain proteinshave the ability to translocate across cell membranes. The shortestinternalizable peptide of a homeodomain protein, Antennapedia, was foundto be the third helix of the protein, from amino acid position 43 to 58(see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634(1996)). Another subsequence, the h (hydrophobic) domain of signalpeptides, was found to have similar cell membrane translocationcharacteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258(1995)).

Examples of peptide sequences which can be linked to a ZFP, forfacilitating uptake of ZFP into cells, include, but are not limited to:an 11 animo acid peptide of the tat protein of HIV; a 20 residue peptidesequence which corresponds to amino acids 84-103 of the p16 protein (seeFahraeus et al., Current Biology 6:84 (1996)); the third helix of the60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol.Chem. 269:10444 (1994)); the h region of a signal peptide such as theKaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); orthe VP22 translocation domain from HSV (Elliot & O'Hare, Cell 88:223-233(1997)). Other suitable chemical moieties that provide enhanced cellularuptake may also be chemically linked to ZFPs.

Toxin molecules also have the ability to transport polypeptides acrosscell membranes. Often, such molecules are composed of at least two parts(called “binary toxins”): a translocation or binding domain orpolypeptide and a separate toxin domain or polypeptide. Typically, thetranslocation domain or polypeptide binds to a cellular receptor, andthen the toxin is transported into the cell. Several bacterial toxins,including Clostridium perfringens iota toxin, diphtheria toxin (DT),Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracistoxin, and pertussis adenylate cyclase (CYA), have been used in attemptsto deliver peptides to the cell cytosol as internal or amino-terminalfusions (Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle etal., Infect. Immun., 61:5147-5156 (1993); Stenmark et al., J. Cell Biol.113:1025-1032 (1991); Donnelly et al., PNAS 90:3530-3534 (1993);Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995);Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNASU.S.A. 89:10277-10281 (1992); and Novak et al., J. Biol. Chem.267:17186-17193 1992)).

Such subsequences can be used to translocate ZFPs across a cellmembrane. ZFPs can be conveniently fused to or derivatized with suchsequences. Typically, the translocation sequence is provided as part ofa fusion protein. Optionally, a linker can be used to link the ZFP andthe translocation sequence. Any suitable linker can be used, e.g., apeptide linker.

The ZFP can also be introduced into an animal cell, preferably amammalian cell, via a liposomes and liposome derivatives such asimmunoliposomes. The term “liposome” refers to vesicles comprised of oneor more concentrically ordered lipid bilayers, which encapsulate anaqueous phase. The aqueous phase typically contains the compound to bedelivered to the cell, i.e., a ZFP. The liposome fuses with the plasmamembrane, thereby releasing the drug into the cytosol. Alternatively,the liposome is phagocytosed or taken up by the cell in a transportvesicle. Once in the endosome or phagosome, the liposome either degradesor fuses with the membrane of the transport vesicle and releases itscontents.

In current methods of drug delivery via liposomes, the liposomeultimately becomes permeable and releases the encapsulated compound (inthis case, a ZFP) at the target tissue or cell. For systemic or tissuespecific delivery, this can be accomplished, for example, in a passivemanner wherein the liposome bilayer degrades over time through theaction of various agents in the body. Alternatively, active drug releaseinvolves using an agent to induce a permeability change in the liposomevesicle. Liposome membranes can be constructed so that they becomedestabilized when the environment becomes acidic near the liposomemembrane (see, e.g., PNAS 84:7851 (1987); Biochemistry 28:908 (1989)).When liposomes are endocytosed by a target cell, for example, theybecome destabilized and release their contents. This destabilization istermed fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basisof many “fusogenic” systems.

Such liposomes typically comprise a ZFP and a lipid component, e.g., aneutral and/or cationic lipid, optionally including areceptor-recognition molecule such as an antibody that binds to apredetermined cell surface receptor or ligand (e.g., an antigen). Avariety of methods are available for preparing liposomes as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO91\17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976);Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys.Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168(1986); Williams et al., PNAS 85:242-246 (1988); Liposomes (Ostro (ed.),1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986);Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: fromPhysics to Applications (1993)). Suitable methods include, for example,sonication, extrusion, high pressure/homogenization, microfluidization,detergent dialysis, calcium-induced fusion of small liposome vesiclesand ether-fusion methods, all of which are well known in the art.

In some instances, liposomes are targeted using targeting moieties thatare specific to a particular cell type, tissue, and the like. Targetingof liposomes using a variety of targeting moieties (e.g., ligands,receptors, and monoclonal antibodies) has been previously described(see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes lipidcomponents, e.g., phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid derivatized bleomycin. Antibody targeted liposomes can beconstructed using, for instance, liposomes which incorporate protein A(see Renneisen et al., J. Biol. Chem., 265:16337-16342 (1990) andLeonetti et al., PNAS 87:2448-2451 (1990).

C. Dosage

For therapeutic applications of ZFPs, the dose administered to a patientshould be sufficient to effect a beneficial therapeutic response in thepatient over time. The dose will be determined by the efficacy and Kd ofthe particular ZFP employed, the nuclear volume of the target cell, andthe condition of the patient, as well as the body weight or surface areaof the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundor vector in a particular patient.

In determining the effective amount of the ZFP to be administered in thetreatment or prophylaxis of disease, the physician evaluates circulatingplasma levels of the ZFP or nucleic acid encoding the ZFP, potential ZFPtoxicities, progression of the disease, and the production of anti-ZFPantibodies. Administration can be accomplished via single or divideddoses.

D. Compositions and Modes of Administration

1. General

ZFPs and the nucleic acids encoding the ZFPs can be administereddirectly to a patient for modulation of gene expression and fortherapeutic or prophylactic applications such as those described infra.In general, and in view of the discussion herein, phrases referring tointroducing a ZFP into an animal or patient can mean that a ZFP or ZFPfusion protein is introduced and/or that a nucleic acid encoding a ZFPof ZFP fusion protein is introduced in a form that can be expressed inthe animal. For example, as described in greater detail in the followingsection, the ZFPs and/or nucleic acids can be used to modulateangiogenesis and in the treatment of ischemia.

Administration of therapeutically effective amounts is by any of theroutes normally used for introducing ZFP into ultimate contact with thetissue to be treated. The ZFPs are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such modulators are available and well known to thoseof skill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions (see,e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)).

The ZFPs, alone or in combination with other suitable components, can bemade into aerosol formulations (i.e., they can be “nebulized”) to beadministered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of the disclosedmethods, compositions can be administered, for example, by intravenousinfusion, orally, topically, intraperitoneally, intravesically orintrathecally. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials.Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

2. Exemplary Angiogenesis Delivery Options

A variety of delivery options are available for the delivery of thepharmaceutical compositions provided herein so as to modulateangiogenesis and thus, for example, the treatment of ischemicconditions. Depending upon the particular application, the compositionscan be targeted to specific areas or tissues of a subject. For example,in some methods, one delivers compositions to specific regions of theheart to treat various disorders such as ischemia. Other treatments, incontrast, involve administering the composition in a general mannerwithout seeking to target delivery to specific regions.

A number of approaches can be utilized to localize the delivery ofagents to particular regions. Certain of these methods involve deliveryto the body lumen or to a tissue (see, e.g., U.S. Pat. Nos. 5,941,868;6,067,988; 6,050,986; and 5,997,509; as well as PCT Publications WO00/25850; WO 00/04928; 99/59666; and 99/38559). Delivery can also beeffectuated by intramyocardial injection or administration. Examples ofsuch approaches include those discussed in U.S. Pat. Nos. 6,086,582;6,045,565; 6,056,969; and 5,997,525; and in PCT Publications WO00/16848; WO 00/18462; WO 00/24452; WO 99/49773 and WO 99/49926. Otheroptions for local delivery include intrapericardial injection (see,e.g., U.S. Pat. Nos. 5,931,810; 5,968,010; and 5,972,013) andperivascular delivery. Various transmyocardial revascular (TMR) channeldelivery approaches can be utilized as well. Many of these methodsutilize a laser to conduct the revascularization. A discussion of suchapproaches is set forth in U.S. Pat. Nos. 5,925,012; 5,976,164;5,993,443; and 5,999,678, for example. Other options includeintraarterial and/or intracoronary delivery, for example coronary arteryinjection (see, e.g., WO 99/29251) and endovascular administration (see,e.g., U.S. Pat. Nos. 6,001,350; 6,066,123; and 6,048,332; and PCTPublications WO 99/31982; WO 99/33500; and WO 00/15285). Thus, forexample, one can inject a composition as described herein directly intothe myocardium.

Additional options for the delivery of compositions to modulateangiogenesis include systemic administration using intravenous orsubcutaneous administration, cardiac chamber access (see, e.g., U.S.Pat. No. 5,924,424) and tissue engineering (U.S. Pat. No. 5,944,754).

Other delivery methods known by those skilled in the art include themethods disclosed in U.S. Pat. Nos. 5,698,531; 5,893,839; 5,797,870;5,693,622; 5,674,722; 5,328,470; and 5,707,969.

IX. Applications

A. General

ZFPs that bind to the target sites disclosed herein, and nucleic acidsencoding them, can be utilized in wide variety of applications,particularly applications involving the modulation of endothelial cellgrowth. Such methods generally involve contacting a target site of anucleic acid within a cell or population of cells with a ZFP that hasbinding specificity for one of the target sites disclosed herein.Methods can be performed in vitro with cell cultures, for example, or invivo.

Certain methods are performed such that modulation involves activationof one or more VEGF genes. Such methods are useful when generation ofadditional endothelial cells is desired, such as in promotingangiogenesis to relieve some type of arterial or vascular blockage or inactivating lymphogenesis or myelopoiesis. Other methods can be conductedto repress endothelial cell growth when such repression provides abeneficial effect, such as inhibiting further vascular development inthe region of a tumor for example.

The ZFPs can also be used for non-therapeutic applications such as inscreening methods to identify agents that activate or repress expressionof a VEGF gene or to detect target nucleic acids containing the targetsequences.

B. Therapeutic Applications

1. Modulation of Angiogenesis

The ZFPs provided herein and the nucleic acids encoding them such as inthe pharmaceutical compositions described supra can be utilized toactivate expression of VEGF genes such that the resulting VEGF proteinscan act as growth factors in activating endothelial cell growth, both incell cultures (i.e., in in vitro applications) and in vivo. Suchactivation can promote useful angiogenesis in which new blood vesselsand capillaries are formed; activation can also promote somatic growthand vascular development and differentiation. Hence, certain methods forpromoting angiogenesis involve introducing a ZFP into an animal. Bindingof the ZFP bearing an activation domain to a gene that modulatesangiogenesis can enhance the process of angiogenesis. Certain methodsinvolve the use of ZFPs such as described herein to bind to target sitesin the VEGF genes. An activation domain fused to the ZFP activates theexpression of one or more VEGF genes.

The ZFPs and nucleic acids can also be useful in bone repair, promotingwound healing and in speeding the healing of gastric and/or duodenalulcers. The ZFPs and nucleic acids can also be utilized to promotedevelopment of the corpus luteum and endometrium, such activation usefulin initiating and/or maintaining pregnancy. In related methods,administration of the ZFPs and nucleic acids also find utility insupporting embryogenesis.

A variety of assays for assessing endothelial cell proliferation andangiogenesis are known. For example, endothelial cell proliferationassays are discussed by Ferrara and Henzel (1989) Nature 380:439-443;Gospodarowicz et al. (1989) Proc. Natl. Acad. Sci. USA, 86: 7311-7315;and Claffey et al. (1995) Biochim. Biophys. Acta. 1246:1-9. The abilityof the ZFPs and/or nucleic acids to promote angiogenesis can beevaluated, for example, in chick chorioallantoic membrane, as discussedby Leung et al. (1989) Science 246:1306-1309. Another option is toconduct assays with rat corneas, as discussed by Rastinejad et al.(1989) Cell 56:345-355. Other assays are disclosed in U.S. Pat. No.5,840,693. In addition, microscopic examination of tissue sections isdisclosed in Example 4 and FIG. 9 can be used as an assay forangiogenesis. Each of these methods are accepted assays of in vivoangiogenesis and the results can also be extrapolated to other systems.

Administration of the ZFPs and/or nucleic acids encoding them can alsobe utilized in applications in which initiation or extension ofvascularization is desirable. Examples of such applications includetherapies following tissue or organ transplantation. Many applicationsinvolve relieving ischemic conditions and various types of blood flowblockage such as that resulting from atherosclerosis. For instance,certain applications involve establishing collateral circulation intissue infarction or arterial stenosis, such as occurs in coronary heartdisease. Stimulation of collateral circulation is also useful intreating deep venous thrombosis, myocardial infarcts, ischaemic limbsand/or postpartum vascular problems.

A variety of assays can be utilized to assess the ability of a ZFP totreat ischemia. For example, several models for studying ischemia areknown. These include, but are not limited to, experimentally induced rathindlimb ischemia (see, e.g., Takeshita, S. et al., Circulation (1998)98: 1261-63; and Takeshita, S. et al. (1994) Circulation 90(#5; partII):228-234), a partially ischemic hindlimb rabbit model (see, e.g.,Hopkins, S. et al., J. Vasc. Surg. (1998) 27: 886-894), and a chronicporcine myocardial ischemia model (see, e.g., Harada, K. et al., Am. J.Physiol. (1996) 270: 886-94; and Hariawala, M. et al., 1996, J. Surg.Res. 63: 77-82). Another assay includes a rabbit model of hindlimbischemia (see, e.g., Takeshita, S. et al., 1994, Circulation 90(#5; partII):228-234).

Neovascularization is also important in fracture repair as the healingprocess involves formation of new blood vessels at the fracture site.Thus, the ZFP and nucleic acids can also be used in the treatment ofbone fractures. Assays for measuring effects of administration areknown. For example, methods for assaying for atherosclerosis arediscussed in PCT Publication WO 00/25805.

Wound treatment is another general type of application in whichadministration of the ZFPs, nucleic acids and compositions disclosedherein find utility. The ZFPs and nucleic acids can be used to treatsignificant wounds such as ulcers, pressure sores and venous ulcers andburns. Examples of such ulcers are those experienced by diabeticpatients. An example of the use of ZFP fusions to promote wound healingis provided in Example 4 and FIG. 9.

The ZFPs and nucleic acids provided herein can also be utilized indiverse surgical applications. For instance, another use is inpreparation of a burn or trauma site to receive a skin graft. Similarly,the compositions can be used to promote the formation of endothelialcells in vascular graft surgery. In such surgeries, compositions can beintroduced at the site of the graft before or during surgery. The ZFPsand nucleic acids can be utilized in plastic surgery, especially inreconstruction of areas burned or otherwise traumatized. Anothersurgical setting in which the compositions can be utilized is inpost-operative wound healing following balloon angioplasty as suchprocedures involve removal and damage to endothelial cells. Treatment insuch instances can utilize compositions containing the ZFP or nucleicacids in combination with a parenteral carrier.

The ZFPs and nucleic acids encoding them also find use in generalsurgery and in treating and repairing cuts and lacerations. In suchapplications, the ZFPs and nucleic acids are used to prevent wounds frombecoming infected. When utilized in topical wound healing, in someinstances the ZFPs or nucleic acids can be administered topically aspart of a solution, spray, cream, gel, ointment or dry powder directlyto the site being treated. Slow release compositions can also beeffective in treating certain types of wounds.

Wound healing assays that can be utilized to asses the utility ofvarious compositions are discussed, for example, by Schilling et al.(1959) Surgery 46:702-710; and Hunt et al. (1967) Surgery 114:302-307.Another wound healing assay is disclosed in Example 4.

The ZFPs and nucleic acids provided herein can also be utilized inpromoting the growth of lymphatic endothelial cells and lymphaticvessels. Additionally, the compositions can be used to stimulatelymphangiogenesis. Thus, for example, the compositions can beadministered to promote regrowth or permeability in lymphatic vessels.This can be useful in treating organ transplant patients and inmitigating the loss of axillary lymphatic vessels that often accompaniessurgery and cancer treatments. Other related applications includetreatment of lymphatic vessel occlusions (e.g., elephantiasis) andlymphangiomas.

Activation of myelopoiesis can also be achieved with certain of thepresent ZFPs and nucleic acids. For example, the compositions can beused to promote the growth of neutrophilic granulocytes. Such treatmentscan be useful in treating patients suffering from diseases such asgranulocytopenia, for example. The production of neutrophilicgranulocytes can be monitored by established microscopic and macroscopicmethods.

Previous experiments showed that the introduction of cDNA encoding asingle mouse VEGF164 isoform in mice led to the formation of permeablevessels which were spontaneously hemorrhagic (Pettersson, A. et al.,2000, Lab. Invest. 80:99-115). Briefly, an adenoviral vector encodingmurine VEGF164 was injected subcutaneously in the ears of mice. VEGF164was shown to induce hemorrhage and extravasation of intravascular dye inthis mouse ear model. When compared to mouse ears similarly treated withan adenovirus encoding murine VEGF164, the ZFP-induced neovasculaturewas not spontaneously hemorrhagic and was not permeable to Evans Bluedye infusion (see Example 6).

2. Repression of VEGF Gene Expression

The compositions provided herein can also be utilized to repressexpression of VEGF genes in a variety of therapeutic applications. Onecommon application is to reduce or inhibit angiogenesis to particularcells or tissues which for therapeutic reasons one wants to destroy.Often such methods involve administration of compositions to preventangiogenic events associated with pathological processes such as tumorgrowth and metastasis. Other pathological processes associated withincreased angiogenesis, particularly in the proliferation of themicrovascular system, include diabetic retinopathy, psoriasis andvarious arthropathies. Thus, certain of the present compositions can beutilized to treat these processes.

C. Non-Therapeutic Applications

The ZFPs and the nucleic acids encoding them can also be utilized invarious non-therapeutic applications. One such application involvesscreening to identify new agents capable of modulating angiogenesis. Inparticular, the ZFPs can be used to identify agents able to modulategene expression, such as the expression of VEGF genes. Such methodsgenerally involve contacting a cell or population of cells with a ZFPthat binds to one of the target sites listed in Tables 2-3 to eitheractivate or repress expression of one or more VEGF genes. The cell(s)are also contacted with a test agent. The level of expression of a VEGFgene is then determined and compared with a baseline level ofexpression. A statistically significant difference in the expressionlevel of the VEGF gene in the test cell(s) with the baseline levelindicates that the test agent is a potential modulator of the expressionof the VEGF gene. For example, if the ZFP activates expression of theVEGF gene, and level of expression in the test cell(s) is lower than thebaseline level, then the evidence indicates that the test agent is arepressor of expression of the VEGF gene. On the other hand, if the ZFPrepresses expression of the VEGF gene, then test agents can be screenedfor potential activators that are able to relieve the repression causedby the ZFP. The method can be varied to introduce a nucleic acidencoding the ZFP, provided the nucleic acid includes the necessaryregulatory sequences for expression in the cells being utilized in theassay. Such methods can be used to identify agents that interact withthe ZFP potentially increasing or decreasing its binding and/or agentscapable of binding elsewhere on the gene, thereby providing either asynergistic or antagonistic effect.

The baseline level of expression refers generally to a value (or rangeof values) against which an experimental or determined value iscompared. Typically, the baseline value is a value determined for acontrol cell or individual treated in parallel and under similarconditions with the cell or individual being tested. The baseline valuecan also be a statistical value (e.g., a mean or average) establishedfor a population of control cells or individuals. In cellular assayssuch as those just described, often the test cell is contacted with aZFP or expresses a ZFP, while the control does not.

A difference is typically considered to be statistically significantwhen the difference is greater then the level of experimental error.Such a difference is also statistically significant if the probabilityof the observed difference occurring by chance (the p-value) is lessthen some predetermined level. Thus, a statistically significantdifference can refer to a p-value that is <0.005, preferably <0.01, andmost preferably <0.001.

In other applications, ZFPs are used in diagnostic methods for sequencespecific detection of target nucleic acid in a sample that includes oneof the target sites provided herein. As an example, ZFPs can be used todetect the presence of particular mRNA species or cDNA in a complexmixtures of mRNAs or cDNAs that includes the target site. Thus, the ZFPscan be used to quantify copy number of VEGF genes in a sample.

A suitable format for performing diagnostic assays employs ZFPs linkedto a domain that allows immobilization of the ZFP on an ELISA plate. Theimmobilized ZFP is contacted with a sample suspected of containing atarget nucleic acid under conditions in which binding can occur.Typically, nucleic acids in the sample are labeled (e.g., in the courseof PCR amplification). Alternatively, unlabeled probes can be detectedusing a second labeled probe. After washing, bound-labeled nucleic acidsare detected.

The following examples are provided solely to illustrate in greaterdetail particular aspects of the disclosed methods and compositions andshould not be construed to be limiting in any way.

Example 1 Regulation of Human VEGF Genes using a Panel of Designed ZincFinger Fusion Proteins I. Introduction

A major question in the study of gene regulation involves the mechanismsby which the cell achieves specific activation of endogenous chromosomalgenes. In addressing this issue, rationally designed components of thetranscriptional machinery can provide powerful tools for testing ourunderstanding of gene regulation (Chatterjee et al. (1995) Nature 374:820-822; Kim et al. (1997) Proc. Natl. Acad. Sci. USA 94: 3616-3620;Klages et al. (1995) Nature 374, 822-823). In particular, artificialtranscription factors—targeted to novel sequences within a given locusand bearing functional domains of the experimenter's choosing—may proveespecially useful since they offer the prospect of completerecapitulation of any given activation process using totally definedcomponents. Artificial transcription factors may also provide practicalbenefits in areas such as medicine and biotechnology.

The DNA binding motif of choice that has emerged for achieving specificrecognition of novel, desired DNA sequences are zinc fingers. Theseinclude the initially-described Cys2-His2 zinc finger, as well asadditional types of zinc finger molecules such as, for example, Cys4zinc fingers and others. See, for example, Rhodes et al. (1993)Scientific American 268: 56-65. Over the past decade, selection anddesign studies have demonstrated the adaptability of this motif and haveyielded simple, powerful strategies for designing zinc finger proteins(ZFPs) that can bind specifically to virtually any DNA sequence. See,for example, Choo et al. (1994) Proc. Natl. Acad. Sci. USA 91:11,163-11,167; Choo et al. (1994) Proc. Natl. Acad. Sci. USA 91:11,168-11,172; Choo et al. (1995) Proc. Natl. Acad. Sci. USA 92: 646(published erratum); Desjarlais et al. (1992) Proc. Natl. Acad. Sci. USA89: 7345-7349; Desjarlais et al. (1992) Proteins 12: 101-104; Desjarlaiset al. (1992) Proteins 13, 272; Desjarlais et al. (1993) Proc. Natl.Acad. Sci. USA 90: 2256-2260; Greisman et al. (1997) Science 275:657-661; Jamieson et al. (1994) Biochemistry 33: 5689-5695; Jamieson etal. (1996) Proc. Natl. Acad. Sci. USA 93: 12,834-12,839; Liu et al.(1997) Proc. Natl. Acad. Sci. USA 94: 5525-5530; Rebar et al. (1994)Science 263: 671-673; Rebar et al. (1996) Meth. Enzymol. 267: 129-149;Segal et al. (1999) Proc. Natl. Acad. Sci. USA 96: 2758-2763. Morerecently, ZFPs with novel, engineered DNA sequence specificities havebegun to be used as artificial transcription factors to regulateendogenous chromosomal genes. See, for example, Bartsevich et al. (2000)Mol. Pharmacol. 58: 1-10; Beerli et al. (2000) Proc. Natl. Acad. Sci.USA 97: 1495-1500; Zhang et al. (2000) J. Biol. Chem. 275:33,850-33,860. See also co-owned PCT WO 00/41566.

The goal of this study was to identify a panel of ZFPs that wouldactivate the endogenous gene for vascular endothelial growth factor A(VEGF-A). VEGF-A is an endothelial cell specific mitogen that isgenerally recognized as the key inducer of new blood vessel growth, bothduring embryogenesis and in adult processes such as wound healing (forrecent reviews see Flamme et al. (1997) Mech. Dev. 63: 51-60; Ferrara(1999) J. Mol. Med. 77: 527-543; Yancopoulos et al. (2000) Nature 407:242-248. Its central roles in both vasculogenesis and angiogenesisapparently necessitate that VEGF-A expression levels be controlled byexquisitely precise regulatory mechanisms. Mouse studies havehighlighted VEGF-A as perhaps the sole example of a gene whosehaploinsufficiency precipitates embryonic lethality (Ferrara et al.(1996) Nature 380: 439-442; Carmeliet et al. (1996) Nature 380: 435-439.Furthermore, several other studies have suggested that proper VEGF-Afunction requires expression of appropriate relative levels of the threemajor splice variants produced by this gene (Carmeliet et al., 1996supra; Carmeliet et al. (1999) Nature Med. 5: 495-502; Grunstein et al.(2000) Mol. Cell. Biol. 20: 7282-7291). A diversity of conditions andtranscription factors have been implicated as inducing VEGF-A expression(Chua et al. (1998) Free Radic. Biol. Med. 25: 891-897; Cohen et al.(1996) J. Biol. Chem. 271: 736-741; Damert et al. (1997) Biochem. J.327: 419-423; Diaz et al. (2000) J. Biol. Chem. 275: 642-650; Ladoux etal. (1994) Biochem. Biophys. Res. Commun. 204: 794-798; Ryuto et al.(1996) J. Biol. Chem. 271: 28,220-28,228; Salimath et al. (2000)Oncogene 19: 3470-3746), of which perhaps the best characterized is thehypoxic response, mediated by HIF-1 (Levy et al. (1995) J. Biol. Chem.270: 13,333-13,340; Liu et al. (1995) Circ. Res. 77: 638-643; Forsytheet al. (1996) Mol. Cell. Biol. 16: 4604-4613; Kimura et al. (2000a)Blood 95: 189-197). Consistent with its highly regulated nature, VEGF-Adysregulation plays a role in a variety of pathological conditions,including tumor growth, diabetic retinopathy, and ischemic heart andlimb diseases. Consequently, VEGF-A would appear to provide anattractive target for both pro- and anti-angiogenic gene therapies usingdesigned artificial transcription factors.

In this example, the inventors have made use of engineered Cys2-His2ZFPs and a knowledge of chromosomal structure to achieve activation ofthe endogenous chromosomal locus containing the gene for VascularEndothelial Growth Factor A (VEGF-A). DNAse I hypersensitivity mappinganalysis was used to identify accessible regions of the VEGF-A locus.This analysis identified four distinct DNAse I-accessible regions inVEGF-A, of which three were present in the HEK293 cells used foractivation studies. Next, eight novel ZFPs were designed to recognize9-bp sequences within each of the three HEK293-specific DNAseI-accessible regions, and their DNA-binding properties werecharacterized. Each designed ZFP bound to its intended target with anapparent Kd of <10 nM. These zinc forgers were then linked to the VP16transcriptional activation domain (Sadowski et al. (1988) Nature 335:563-564), and tested for their capacity to activate transcription ofboth the endogenous VEGF-A gene and transiently transfected nativereporter constructs containing ˜3 kb of the VEGF-A promoter. The resultsindicate that each of the designed ZFP-VP16 fusions activates both theendogenous VEGF-A locus and the native reporter constructs. The designedZFPs were also linked to the activation domain from the p65 subunit ofNFκB (Ruben et al. (1991) Science 251: 1490-1493; published erratumappears in Science (1991) 254: 11), and tested for the capacity toactivate transcription of endogenous VEGF-A both alone and in certaincombinations with VP 16-linked ZFPs.

This strategy has yielded eight distinct ZFPs, targeted to seven 9-bpsites, that activate VEGF-A expression. For certain ZFPs, linkage toactivation domains from either VP16 or p65 provides differing levelsactivation, dependent on the chromosomal site that is targeted.Furthermore, when certain combinations of VP16- and p65-linked ZFPs(targeted to distinct chromosomal sites) are cotransfected, the observedVEGF-A activation is more than additive relative to the activationlevels of the individual ZFPs. Finally, it is disclosed that the levelsof activation achieved by these engineered transcription factors exceedVEGF-A levels attained during the hypoxic response and that the relativeproportions of VEGF-A splice variants produced by this activationclosely approximates those normally observed in these cells.

II. Experimental Procedures

Cell lines and cell culture. Immortalized cell lines used in thesestudies (HEK 293, Hep3B, and H9c2(2-1)) were obtained from the AmericanType Culture Collection, and human primary skeletal muscle cells wereobtained from Clonetics. Each line was maintained essentially asrecommended by the suppliers. Rat primary cardiac myocytes wererecovered from the hearts of day 1 neonatal Wistar-Han rats (CharlesRiver) via dissociation with a solution of 115 U/ml type II collagenaseand 0.08% pancreatin. They were then purified on a discontinuous Percollgradient, resuspended in a plating medium containing 15% serum, andplated on gelatin-coated plates for 24 hr. Cells were then maintained ina serum-free medium for 24 to 48 hrs prior to use in DNase I mappingstudies.

Mapping of DNase I-accessible chromatin regions in the VEGF-A locus.Nuclei were isolated and treated with DNase I (Worthington) essentiallyas described by Zhang et al., supra, except that DNase I digestions werefor 1.5 min at 22° C. and the concentrations of DNase I used were asindicated in the legend to FIG. 1. Genomic DNA isolation, restrictionenzyme digestion, and Southern blot analysis were then performedessentially as described by Zhang et al., supra, except that enzymes andprobes were as indicated in the legend to FIG. 1. See also co-owned U.S.Patent Application Ser. Nos. 60/200,590 and 60/228,556 for additionaldisclosure regarding identification of accessible regions in cellularchromatin.

Assembly of ZFP-encoding polynucleotides; synthesis, purification andbinding analysis of zinc finger proteins.—Genes encoding VEGF-A-targetedZFPs were assembled, cloned and purified as previously described. See,for example, Zhang et al., supra; WO 00/41566; and WO 00/42219. Briefly,oligonucleotides encoding α-helix and β-sheet regions of eachthree-finger protein were assembled using PCR (FIGS. 2A and 2B), andeach resultant ZFP gene was cloned into the pMal-c2 plasmid (New EnglandBiolabs, Beverly, Mass.) as a fusion with DNA encoding the maltosebinding protein. Maltose binding protein-ZFP fusions were then expressedand affinity purified using an amylose resin (New England Biolabs,Beverly, Mass.).

Binding studies were performed essentially as described (Zhang et al.,supra; WO 00/41566; and WO 00/42219) except that the binding reactionscontained 10 pM of labeled target site and the buffer composition was asfollows: 17 mM Tris, 170 mM KCl, 1.7 mM MgCl2, 3.5 mM DTT, 0.01-0.033 mMZnCl2, 15% glycerol, 300 μg/ml bovine serum albumin, 0.03% IGEPAL. Inaddition, ZFP concentrations for these studies were determined directlyby measuring the DNA-binding activity of each ZFP preparation usingconditions under which binding is essentially stoichiometric (ZFP andtarget site concentrations of >100 nM). Using this modified protocol,the SP1 zinc finger protein exhibits a significantly higher affinity forits target site than was determined in previous studies (Zhang et al.,supra) and it is likely that both the use of activity-based estimates ofZFP concentration and the new binding buffer in these studiescontributed to the difference in apparent Kds.

Construction of Zinc Finger Fusion Proteins. VEGF-A-targeted zincfingers were assembled in an SP1 backbone and cloned into the pcDNA3mammalian expression vector (Invitrogen, Carlsbad, Calif.) as describedpreviously (Zhang et al., supra; WO 00/41566; and WO 00/42219). A CMVpromoter was used to drive the expression of all the ZFPs in mammaliancells. All ZFP constructs contained an N-terminal nuclear localizationsignal (Pro-Lys-Lys-Lys-Arg-Lys-Val, SEQ ID NO:224) from SV40 large Tantigen, a Zinc Finger DNA-binding domain, an activation domain, and aFLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, SEQ ID NO:225). ZFP-VP16fusions contained the herpes simplex virus VP16 activation domain fromamino acid 413 to 490 (Sadowski et al., supra; Zhang et al, supra; WO00/41566; and WO 00/42219). ZFP-p65 fusions contained the human NF-κBtranscription factor p65 subunit (amino acid 288-548) as the activationdomain (Ruben et al., supra).

Assay for the activity of ZFP fusions for human VEGF-A reporteractivation. The effect of ZFPs on human VEGF-A promoter activity wasmeasured using a luciferase reporter construct containing the humanVEGF-A promoter. The human VEGF-A luciferase reporter construct pGLPVFHwas made by inserting a genomic DNA fragment containing 3318 base pairsof the human VEGF-A promoter and its flanking sequences (nt −2279 to+1039 with respect to the transcription start site) into the multiplecloning site region of the pGL3 vector (Promega, Madison, Wis.) betweenthe KpnI and NcoI sites. The translation start codon ATG of the VEGF-Agene was directly fused with the luciferase gene in this construct.Human embryonic kidney cells (HEK 293) were grown in DMEM (Dulbecco'smodified Eagle's medium), supplemented with 10% fetal bovine serum, in a5% CO₂ incubator at 37° C. Cells were plated in 24-well plates at adensity of 160,000 cells per well a day before transfection. Thereporter construct and ZFP-VP16 fusion plasmid were co-transfected intothe cells via LipofectAMINE reagent (Gibco Life Technologies, Rockville,Md.) according to manufacturer's recommendations, using 1.5 μlLipofectAMINE reagent, 260 ng of the VEGF-A reporter construct, 30 ng ofplasmid DNA encoding ZFP-VP16, and 10 ng of control pRL-CMV plasmid(Promega, Madison, Wis.). Medium was removed and replaced with freshmedium 16 hours after transfection. Forty hours after transfection,medium was removed, and the cells were harvested and assayed forluciferase reporter activity using the Dual-luciferase Assay System(Promega, Madison, Wis.) according to manufacture's protocol.

Assays for the activity of ZFP fusions on the endogenous VEGF-A gene inhuman cells by transient transfection. Human embryonic kidney cells (HEK293) were grown in DMEM (Dulbecco's modified Eagle's medium),supplemented with 10% fetal bovine serum, in a 5% CO2 incubator at 37°C. Cells were plated in 24-well plates at a density of 160,000 cells perwell. A day later, plasmids encoding ZFP-VP16 fusions were transfectedinto the cells using LipofectAMINE reagent (Gibco Life Technologies,Rockville, Md.) according to manufacture's recommendations, using 1.5 μlLipofectAMINE reagent and 0.3 μg ZFP plasmid DNA per well. Cells aretransfected at 50% confluence or 90% confluence, or any integralpercentage therebetween. Medium was removed and replaced with freshmedium 16 hours after transfection. Forty hours after transfection, theculture medium and the cells were harvested and assayed for VEGF-Aexpression. VEGF-A protein content in the culture medium was assayedusing a human VEGF ELISA kit (R&D Systems, Minneapolis, Minn.) accordingto manufacture's protocol.

For western analysis of ZFP protein expression, cells were lysed withLaemmli Sample Loading Buffer and the lysates were analyzed by a 10%polyacrylamide gel electrophoresis (BioRad, Hercules, Calif.) followedby western blotting using anti-FLAG antibody (Sigma, St. Louis, Mo.)which recognizes the FLAG epitope tag of the designed ZFPs. The westernblots were visualized by ECL (Amersham Phamacia Biotech, Piscataway,N.J.) as described previously (Zhang et al., supra).

For quantitative RT-PCR analysis of VEGF mRNA level, the cells werelysed and total RNA was prepared using the RNeasy Total RNA IsolationKit with in-column DNase treatment, according to the manufacturer'sinstructions (Qiagen, Valencia, Calif.). Twenty five ng RNA were used inreal-time quantitative RT-PCR analysis using Taqman chemistry on an ABI7700 SDS machine (Perkin Elmer Applied Biosystems, Foster City, Calif.)as described previously (Zhang et al., supra). Briefly, reversetranscription was performed at 48° C. for 30 min using MultiscribeReverse Transcriptase (PE BioSystems, Foster City, Calif.). Following a10 min denaturation at 95° C., PCR amplification using AmpliGold DNApolymerase was conducted for 40 cycles at 95° C. for 15 seconds and at60° C. for 1 minute. The results were analyzed using SDS version 1.6.3software (PE BioSystems, Foster City, Calif.). The primers and probesused for Taqman analysis are listed in Table 5. These primers and probesrecognize all known splice variants of the human VEGF-A gene, but do notdistinguish among them.

TABLE 5 Nucleotide sequences of primers and probes used for Taqmananalysis Sequence SEQ ID NO. VEGF-A forward primer5′-GTGCATTGGAGCCTTGCCTTG-3′ 226 VEGF-A reverse primer5′-ACTCGATGTCATCAGGGTACTC-3′ 227 VEGF-A Taqman Probe5′-FAM-CAGTAGCTGCGCTGATAGACATCCA- 228 TAMRA-3′ GAPDH forward primer5′-CCATGTTCGTCATGGGTGTGA-3′ 229 GAPDH reverse primer5′-CATGGACTGTGGTCATGAGT-3′ 230 GAPDH Taqman Probe5′-FAM-TCCTGCACCACCAACTGCTTAGCA- 231 TAMRA-3′ VP16-FLAG forward5′-CATGACGATTTCGATCTGGA-3′ 232 primer VP16-FLAG reverse5′-CTACTTGTCATCGTCGTCCTTG-3′ 233 primer VP16-FLAG Taqman5′-FAM-ATCGGTAAACATCTGCTCAAACTCGA- 234 Probe TAMRA-3′ Abbreviations:FAM: aminofluorescein; TAMRA: tetramethylrhodamine

RNA blot analysis of VEGF mRNA. HEK 293 cells were grown in 150 mmdishes and transfected with a pcDNA3 vector control or a ZFP-encodingplasmid, using LipofectAMINE reagent (Gibco Life Technologies,Rockville, Md.) according to the manufacture's recommendations. Cellsand conditioned media were harvested forty hours after transfection.Total RNA was extracted from the cells using Trizol reagent (Gibco LifeTechnologies, Rockville, Md.) followed by RNeasy total RNA isolationmidi-prep system (Qiagen, Valencia, Calif.). For RNA blots, total RNAsamples (30 μg) were resolved on a 1.2% formaldehyde agarose gel andblotted onto a Nytran SuperCharge membrane (Schleicher & Schnell, Keene,N.H.). The membrane was hybridized to ³²P-labeled human VEGF-A165antisense riboprobe at 68° C. in Ultrahyb™ hybridization buffer (Ambion,Austin, Tex.). After washing with 0.1×SSC, 0.1% SDS at 68° C., themembrane was exposed to film. The same membrane was stripped by boilingin 0.1% SDS and re-hybridized with a human β-actin antisense riboprobe.

Analysis of splice variants of VEGF-A mRNA—To detect the multiple splicevariants of VEGF-A mRNA, total RNA samples (0.5 μg) were subjected to a20-cycle RT-PCR reaction using Titan™ one-tube RT-PCR system (RocheMolecular Biochemicals, Indianapolis, Ind.). The primers used were5′-ATGAACTTTCTGCTGTCTTGGGTGCATT-3′ (SEQ ID NO:235), and5′-TCACCGCCTCGGCTTGTCACAT-3′ (SEQ ID NO:236). The PCR products wereresolved on a 3% Nusieve 3:1 agarose gel (FMC, Rockland, Me.), blottedonto a Nytran SuperCharge membrane (Schleicher & Schuell, Keene, N.H.),and analyzed by Southern hybridization using a ³²P-labeled humanVEGF-A165 antisense riboprobe. The expected PCR product sizes forVEGF-189, VEGF-165 and VEGF-120 were 630, 576, and 444 bp, respectively.

III. Results

Constitutive and Cell-Specific Regions of Accessible ChromatinAssociated with VEGF-A.—Chromatin mapping studies encompassed a varietyof cell types from human and rat, including both tumor lines and primarycells. The scope of this survey, as well as choice of cell types, wasmotivated by the goal of developing candidate ZFPs for a variety of pro-and anti-angiogenic gene therapies. An initial goal was to identify anyconstitutive accessible chromatin regions in the VEGF-A promoter, aswell as those specific to medically relevant target tissues and modelorganisms. Accordingly, DNAse hypersensitivity analysis was conducted onchromatin from a number of different primary cells and cell lines. Theresults of this analysis indicate the existence of four distincthypersensitive regions in or near the VEGF-A gene. Two of these,centered on approximately by −550 and +1 of the VEGF-A promoter, wereinvariably observed in every cell type tested. FIG. 1 shows typicalexperimental results identifying these regions in both human (FIG. 1A)and rat (FIG. 1B). Both regions appear as doublets when viewed at higherresolution (FIGS. 1A and 1C). The discovery of an open region at ‘+1’was somewhat expected, as hypersensitive regions are often observed inthe vicinity of sites for transcription initiation (see, e.g., Gross etal. (1988) Ann. Rev. Biochem. 57: 159-197), and this region furthercontains several conserved regulatory elements which have been shown tobe important for VEGF-A activation, including targets for SP1 and AP-2.Milanini et al. (1998) J. Biol. Chem. 273: 18,165-18,172. Theobservation of accessible chromatin in the ‘−550’ region (in both humanand rat chromatin) was somewhat more surprising, as no regulatoryelements have thus far been mapped to this area of the VEGF-A promoter.However, it is noted that this region of the VEGFA promoter exhibits ahigh degree of sequence conservation among humans, mouse and rat (FIG.1F, gray trace), which becomes more pronounced when conservation isassessed in terms of regulatory element-sized sequence blocks, (FIG. 1F,black trace), and that the DNAse I hypersensitivity of this region (aswell as that of the ‘+1’ region) is also conserved between rat andhuman. Indeed, analysis of a construct comprising a DNA fragmentcontaining this region, fused to a SV40 basal promoter driving aluciferase reporter gene, indicates that this region mediates cis-actingregulatory functions. The possibility that the −550 region wasinherently more susceptible to DNAse I digestion was excluded byperforming a control mapping study using purified naked genomic DNA,that indicated no enhanced cutting of this region by DNAse I.

In addition to the constitutive hypersensitive regions found at −550 and+1, two other stretches of accessible chromatin, apparent in only asubset of cell types tested, were identified. One of these regionsencompassed approximately 300 by centered on the hypoxia responseelement (HRE) of VEGF-A, and was observed in human primary skeletalmuscle cells (FIG. 1C) and in the Hep3B cell line. In contrast, thissite was clearly not observed in HEK293 cells (FIG. 1C). The hypoxiaresponse element encompasses a region of enhanced sequence conservationin the VEGF-A promoter (FIG. 1F), and has been shown to contain severalconserved regulatory elements that are required for induction of VEGF-Aby hypoxia, including a binding site for HIF-1. Ikeda et al. (1995) J.Biol. Chem. 270: 19,761-19,766; Liu et al. (1995) supra; Grant et al.(2000) Biochemistry 39: 8187-8192. Finally, a hypersensitive region wasobserved, approximately 500 by downstream of the transcription startsite, in HEK293 cells (FIG. 1D), primary skeletal muscle cells, and ratprimary cardiac myocytes (FIG. 1B). Interestingly, this region containsa putative SP1 regulatory element and is adjacent to an alternativetranscription start site (FIG. 1F). Akiri et al. (1998) Oncogene 17:227-236. This region also displayed the capacity to activate theexpression of a reporter gene in cis. In summary, the inventors haveidentified three regions accessible to DNAse I in HEK293 cells, centeredat approximately by −550, +1, and +500 relative to the transcriptionalstart site of VEGF-A.

Design and Biochemical Characterization of ZFPs targeted to openchromatin regions of VEGF-A. ZFPs that bind to sequences containedwithin each of the hypersensitive regions described above were designed.Design and selection studies of zinc finger-DNA recognition have yieldeda diverse collection of fingers with characterized tripletspecificities. See, for example, Elrod-Erickson et al. (1998) Structure6: 451-464 and references cited supra. Collectively, these fingersprovide a directory of triplet-binding modules that can, under certainconditions, be mixed and matched to obtain multifinger proteins withdesired binding properties. Using this approach, a series of ZFPstargeted to sites within the ‘−550’, ‘+1’ and ‘+500’ open chromatinregions observed in 293 cells (FIG. 2D) were designed, and genesencoding these ZFPs were assembled. Genes encoding two control ZFPs,targeted to sites outside of the hypersensitive regions, were alsoassembled. Designs for these ZFPs are shown in Tables 3, 4 and 6. TheZFPs were named according to their target location relative to thetranscription start site of VEGF-A. Thus the first base in the targetsequences of VZ−475 and VZ+590 lie 475 base pairs upstream and 590nucleotides downstream, respectively, of the principal transcriptioninitiation site of the VEGF-A gene. One of these ZFPs has two targetsites in this region and so has been given a complex name to reflectthis fact (VZ+42/+530). Where multiple ZFPs are targeted to a givensequence, this is indicated by the use of a small letter suffix at theend of each name to distinguish between alternate ZFP designs.

Using previously described methods (see, for example, co-owned WO00/41566 and WO 00/42219, and Zhang et al., supra), genes encoding theZFP designs were assembled and each of the encoded proteins wasexpressed in recombinant form (FIGS. 2A-2C). The DNA-binding affinity ofeach of the ZFPs was then characterized using a gel shift assay. All ofthe designed ZFPs exhibited apparent K_(d)s for their intended DNAtargets that were in the nanomolar range. For comparison, under theseconditions SP1 exhibited an apparent K_(d) of 0.25 nM for its DNAtarget. These studies demonstrated that each designed ZFP recognizes itstarget site with high affinity.

Activation of the human VEGF-A gene promoter by ZFPs. Each of thedesigned ZFPs was fused to the minimal activation domain of the VP16transcription factor and tested for its ability to activate a reportergene under the control of a VEGF-A promoter. Fusion constructs alsocontained a N-terminal nuclear localization sequence and a C-terminalFLAG epitope tag. The reporter plasmid was constructed to contain afirefly luciferase gene under the control of human VEGF-A promoter. Whentransiently cotransfected into cells with the reporter plasmid, all ofthe designed ZFP-VP16 fusions were able to activate the reporter (FIG.3A). The range of activation varied between 3- to 15-fold. Theactivation was ZFP-dependent; since a Green Fluorescent Protein-VP16fusion was unable to activate the reporter. See also FIG. 12. Theseresults showed that all of the designed ZFPs were active on anextrachromosomal DNA template.

Transcriptional activation of an endogenous human VEGF-A gene usingZFPs. To test whether these ZFP-VP16 fusions were also active inregulating VEGF-A gene transcription from the endogenous chromosomallocus, the designed ZFP-VP16 fusions were transiently transfected intoHEK293 cells, and their effect on endogenous VEGF-A gene expression wasanalyzed. Human embryonic kidney cells (HEK 293) produced relatively lowlevels of VEGF-A in the absence of any ZFP constructs. As shown in FIG.3C, expression of the ZFP-VP16 fusions designed as described aboveresulted in the secretion of elevated levels VEGF-A into the culturemedium, as determined by ELISA. The range of activation varied between2- to 15-fold, with ZFP VZ+434b the most active. The increased VEGF-Aprotein production induced by ZFP was correlated with a 2- to 10-foldincrease in the level of VEGF-A mRNA as determined by quantitative PCR(FIG. 3D). The various ZFP-VP16 fusions activated human VEGF-A mRNAtranscription with varying abilities, with ZFP VZ+434b being the mostactive. The different ZFP fusions were found to be expressed to similarlevels, as determined by western blotting for protein levels (FIG. 3E)and by Taqman for mRNA expression (FIG. 3F). See also FIGS. 10 and 13(for VEGF protein levels induced by additional ZFP constructs) and FIG.11 (for a corresponding analysis of VEGF mRNA levels).

The behavior of ZFPs targeted to the accessible regions of the VEGF Agene was compared with that of ZFPs targeted to non-accessible regions.Representative data from these studies are shown in FIG. 4. Whereas allfour ZFPs tested activated the extrachromosomal reporter construct toapproximately equal levels, a clear discrepancy was seen regardingactivity against the endogenous VEGF-A gene. The accessible-regiontargeted ZFPs activated VEGF-A by factors of 4 to 5 while the ZFPstargeted to sites outside of the accessible regions showed noappreciable increase in VEGF-A expression level. Thus, in certain cases,it can be advantageous to target a designed ZFP to a binding sitepresent in an accessible region of cellular chromatin.

Activation of a human VEGF-A gene by ZFPs fused with differentactivation domains. To achieve a higher level of VEGF-A activation byZFPs in human cells, the performance of other activation domains wastested and compared to that achieved by VP16. It was found that use ofthe activation domain from the p65 subunit of NF-κB provided higherlevels of activation, when tested using several of the designed ZFPs(FIG. 5). In this experiment, some ZFP-p65 fusions, for example the ZFPVZ+434b, induced levels of VEGF-A protein that were 3- to 4-fold higherthan those induced by ZFP-VP16 fusions (FIG. 5C), even though theZFP-p65 fusion proteins and the ZFP-VP16 fusion proteins were expressedand accumulated in cells to similar levels (FIG. 5B). The higher levelof VEGF-A protein production induced by the ZFP-p65 fusion wasconsistent with a higher level of VEGF-A mRNA transcription, asdetermined by Taqman analysis (FIG. 5D). Interestingly, for some ZFPs,such as VZ-8, the p65 fusions displayed activities similar to those ofthe VP16 fusions. Therefore, it seems that VP16 and p65 have differenttarget site-dependent activation mechanisms.

Synergy between ZFPs in the activation of a human VEGF-A gene. Theavailability of a set of activating ZFPs targeted to diverse regions ofthe VEGF gene promoter, and fused to different activation domains,provided an opportunity to investigate whether combinations of ZFPs withdifferent activation domains could achieve synergistic control of VEGFgene transcription. To test the possibility, various ZFP-VP16 andZFP-p65 fusions were cotransfected into human HEK293 cells, and VEGFlevels were determined by ELISA and Taqman. As shown in FIG. 6, the ZFPsVZ+434b−VP16 and VZ−573a−p65 activated VEGF production in 293 cells by8- and 6-fold respectively. However, when these two fusions wereco-transfected at a 1:1 ratio into the cells, the level of VEGF geneactivation (30-fold) was more than additive, compared to the levelsinduced by each individual ZFP. A similar synergy between the ZFPsVZ+434b−p65 and VZ−573a−VP16 was also observed.

Comparison of VEGF induction by ZFPs with levels of VEGF-A induced byhypoxia.—In order to assess the magnitude of activation of VEGF-Aexpression by ZFP fusions, with respect to activation of VEGF by aphysiologically relevant processes, VEGF levels induced by ZFP fusionswere compared with those induced by hypoxia. For many of the ZFP fusionstested, for example ZFP VZ+434b, the ZFPs were capable of activatingVEGF-A expression to a level higher than that induced by hypoxia. Asshown in FIG. 7, HEK293 cells grown under hypoxic conditions had asteady-state VEGF-A mRNA level that was 5-fold higher than that in cellsgrown in normoxic conditions (FIG. 7B), and accumulated VEGF-A proteinto nearly 400 pg/ml in the culture medium (a 10-fold increase, FIG. 7A).ZFP VZ+434b fused to a p65 activation domain induced expression of theVEGF gene to levels 5- to 10-fold greater than that induced by hypoxia,as evidenced by an accumulation of VEGF protein in culture medium tonearly 4000 pg/ml (FIG. 7A) and a 20-fold increase in VEGF mRNA level(FIG. 7B). This observation was also confirmed by RNA blot analysis(FIG. 7C).

Activation of multiple VEGF splice variants using a single ZFP. Severalsplice variants of human VEGF-A mRNA have been reported, each onecomprising a specific exon addition. Ferrara (1999) supra. The majorVEGF mRNA splice variants produce polypeptides with 121, 165, and 189and 206 amino acids, although VEGF206 is rarely expressed and has beendetected only in fetal liver. Because the designed ZFPs activated genetranscription from the natural chromosomal promoter (see supra), theywould be expected to activate all of the different VEGF-A transcriptsequally, preserving their relative proportions. To confirm this notion,the ability of the ZFP VZ+434b to activate multiple transcripts from thesame promoter was analyzed. To distinguish the splice variants, RT-PCRwas performed using primers flanking the regions of differentialsplicing, such that a distinct PCR product is produced for each splicevariant. The PCR products were then analyzed by Southern hybridizationusing a VEGF165 probe. Three splice variants, VEGF-A-189, VEGF-A-165,and VEGF-A-121 were detected in 293 cells, with VEGF-A-165 being thepredominant form. As demonstrated in FIG. 7D, the introduction of asingle ZFP, VZ+434b, as either a VP16 or a p65 fusion, resulted in aproportional increase in levels of all of the splice variants.

IV. Discussion

This example demonstrates the successful design of a panel of ZFPscapable of activating the endogenous human VEGF-A gene from diversetarget sites within its promoter. The experimental approach incorporatedinformation regarding the chromatin structure of the VEGF-A locus,since, in certain circumstances, information regarding chromatinstructure may be used in combination with ZFP design principles toprovide efficient means for identifying artificial transcription factorscapable of specifically regulating endogenous genes. Several of thedesigned ZFPs disclosed herein are potent activators, yielding VEGF-Alevels exceeding those observed during induction by hypoxia. While anunderstanding of mechanism is not required for the practice of thedisclosed methods and/or use of the disclosed compositions, it ispossible that designed ZFP fusions targeted to DNAse I accessibleregions may act synergistically with the natural transcription factorswhich presumably bind to these regions.

The panel of artificial transcription factors disclosed herein, whichare targeted to diverse sites in the VEGF-A gene, provides a unique toolfor assessing the structural determinants of transcriptional activationat an endogenous locus, and several of the results reflect possibletranscriptional effects of chromatin structure. For example, differentmembers of the panel of ZFPs exhibit different patterns of activation ofthe endogenous VEGF-A locus, compared to an extrachromosomal promoterreporter construct (compare FIGS. 3B and 3C). Furthermore, the abilityof the p65 activation domain to outperform VP16 varies in aposition-dependent manner, with relative activation levels varying overa factor of three, depending on the location of the ZFP target withinthe VEGF-A locus (FIG. 5C). These effects could reflect limitations onthe capacity of designed transcription factors to activate an endogenouslocus resulting from the structural context imposed by chromatin,possibly coupled with the binding of other regulatory proteins. Theyalso reemphasize the idea that different activation domains havedistinct regulatory and steric requirements for optimal performance.

The ability to generate a panel of factors targeted to an endogenouslocus also provides practical advantages in a variety of applications.In studying the effects of up- or down-regulation of a target locus, forexample, conclusions regarding gene function will be strongest if agiven effect is observed repeatedly using a number of differentregulators. In addition, for potential medical uses, the availability ofmultiple ZFP candidates provides a greater likelihood of obtaining alead which yields optimal benefits with minimal side effects. Additionalpossibilities for use of these proteins are in the study oftranscriptional regulation. For example, the ability to target multipleactivation domains to arbitrary sites in the same locus has clearapplications in the study of synergy. In this respect, greater thanadditive effects on VEGF expression, mediated by cotransfected VP 16-and p65 activation domain-bearing ZFPs, has been disclosed herein. It islikely that, by using larger combinations of appropriately targetedfunctional domains, designed ZFPs may offer the prospect for totalreconstitution of activation processes using completely definedcomponents.

An additional observation of the studies disclosed herein is that ZFPsfused with the NF-κB p65 subunit (Ruben et al., supra) activate theendogenous VEGF gene as well as or better than the 78 amino acidactivation subdomain of the Herpes Simplex Virus VP16 protein (Sadowskiet al., supra). Although both VP16 and NF-κB p65 are strong acidicactivation domains and share certain functional features; for example,recruitment of the ARC/DRIP complex and facilitated assembly of apreinitiation complex on promoter DNA (Naar et al. (1999) Nature 398:828-832; Rachez et al. (1999) Nature 398: 824-828); differences in theirtransactivation mechanism have been reported. For example, the histoneacetyltransferase activity of p300 has been demonstrated to be necessaryfor activation by NF-κB, but less essential for activation by VP16.Kraus et al. (1999) Mol. Cell. Biol. 19: 8123-8135; Li et al. (2000)Mol. Cell. Biol. 20: 2031-2042. It is possible that the localavailability of p300 or other coactivators in the endogenous VEGF locusmay account for the observed differences.

Finally, the designed ZFPs disclosed herein upregulate each major splicevariant of VEGF-A, in proportions similar to those observed underphysiological conditions (e.g., hypoxia). This is important becauserecent studies suggest that proper isoform balance is crucial for VEGF-Afunction. Carmeliet et al. (1996) supra; Carmeliet et al. (1999) supra;Grunstein et al. (2000) supra. In particular, the 165, 189 and 206 aminoacid isoforms of VEGF-A have increasingly stronger heparin-bindingdomains, which are involved in presentation to VEGF receptors and inbinding to the extracellular matrix. Heparin-binding ability is acritical determinant of VEGF-A potency, resulting in differentbiological activities for different isoforms. Currently, most VEGF-Agene therapy trials involve the application of just a single VEGF-Asplice variant cDNA or protein isoform. Isner et al. (1996) Lancet 348:370-374; Esakof et al. (1999) Hum. Gene Ther. 10: 2307-2314; Rosengartet al. (1999a) Ann. Surg. 230: 466-470, discussion 470-472; Rosengart etal. (1999b) Circulation 100: 468-474; Hendel et al. (2000) Circulation101: 118-121. It has been suggested that an ideal gene therapy agentshould be able to recapitulate natural ratios of various differentVEGF-A isoforms. Activation of VEGF-A using the designed ZFPs disclosedherein therefore offers advantages in this regard, providing keycomponents of pro-angiogenic gene therapy agents.

Example 2 Activation of an Endogenous Mouse VEGF Gene

The sequence of the murine VEGF gene (GenBank Accession Number U41383)was searched for ZFP target sites and a ZFP, denoted VG10A/8A, wasdesigned to bind to a site between 56 and 73 nucleotides downstream ofthe transcriptional startsite. The sequence of this target site is5′-TGAGCGGCGGCAGCGGAG (SEQ ID NO:237). The six-finger ZFP designed tobind this target site has the following amino acid sequences in therecognition helices (proceeding in an N-terminal to C-terminaldirection): RSDNLAR (SEQ ID NO:35); RSDELQR (SEQ ID NO:159); QSGSLTR(SEQ ID NO:57); RSDELTR (SEQ ID NO:122); RSDELSR (SEQ ID NO:238) andQSGHLTK (SEQ ID NO:239). This six-finger binding domain was fused to aVP16 activation domain, according to methods described in Example 1. Aplasmid encoding this ZFP fusion was co-transfected into mouse cellswith a reporter gene under the control of the murine VEGF promoter, anda 29-fold activation of reporter gene activity was observed.

This ZFP fusion construct was also injected into mouse skeletal muscleto test for activation of the endogenous VEGF gene. One hundredmicroliters of a 1 mg/ml solution of plasmid DNA was injected into twoseparate sites in the quadriceps muscle of a live mouse. Thecontralateral quadriceps muscle was subjected to two control injections,of a plasmid lacking ZFP-encoding sequences, at sites in the musclesimilar to those receiving the experimental injections. Injectionneedles were marked to assure similar depths of injection, and sites ofinjection were marked with India Ink.

Three days after injection, identically-sized tissue samples wereharvested from the marked injection sites by punch biopsy. Proteins wereextracted from the tissue samples and separated by gel electrophoresis.The gel was blotted and the blot was probed with an anti-mouse VEGFantibody (R&D Systems, Minneapolis, Minn.). Results are shown in FIG. 8.The results indicate that production of VEGF is enhanced in mouse musclethat has been injected with a plasmid encoding a ZFP-VP16 fusion, andthat the enhancement of VEGF expression is not simply due to injectionper se.

Example 3 Activation of an Endogenous Rat VEGF Gene

Experiments similar to those described in Example 2 were performed onrats, using ZFPs targeted to sites in the rat VEGF gene, certain ofwhich are homologous to ZFP target sites in the human VEGF-A gene andwere shown to activate the human VEGF-A gene (see Example 1, supra).Sequences of the target sites and recognition helices of these ZFPs areprovided in Table 7. ZFP-VP16 fusions were injected into rat skeletalmuscle, similar to the mouse injections described in Example 2. Analysisby immunoblotting, using an anti-rat VEGF antibody (R&D Systems,Minneapolis, Minn.), showed that all of the ZFP fusions activated ratVEGF production, and that BV012A-11A, BVO14A-13B, and VOP 32B induced amarked increase in VEGF expression, between 5- and 10-fold.

Example 4 Stimulation of Wound Healing and Angiogenesis in Mice UsingVEGF-Targeted ZFP Fusions

In this example, punch biopsy wounds were made in both quadricepsmuscles of a mouse. A plasmid encoding the VG10A/8A ZFP-VP16 fusionunder the transcriptional control of a CMV promoter (as used in Example2, supra) was injected into the periphery of one of the wounds, and acontrol plasmid, lacking sequences encoding a ZFP binding domain, wasinjected into the contralateral wound. After three days, tissue wasexcised, and hematoxylin & eosin-stained thin sections were examinedmicroscopically. Results are shown in FIG. 9. FIG. 9B shows alow-magnification image in which margins of ingrowing tissue areapparent beneath the blood clot covering the wound (clot at top left,margins of ingrowing tissue indicated by arrowheads). In comparison, noingrowing tissue is seen beneath the clot in the control-injected tissueshown in FIG. 9A. FIGS. 9D and 9F show high-magnification images whichreveal increased vascularization in the ZFP-injected tissue, evidencedby a larger number of capillary sections and red blood cells, comparedto the control-injected tissue shown in FIGS. 9C and 9E.

The results of this experiment indicate that activation of the VEGF geneby targeted ZFP fusions leads to faster wound healing and increasedvascularization.

Example 5 Regulation of Multiple Human VEGF Genes Using a Single ZFPFusion Protein

In this example, the ability of a single ZFP fusion to regulate aplurality of human VEGF genes was tested. As shown in Table 2, the VOP28A and VOP 30A ZFPs have target sites in both the VEGF-A and VEGF-Cgenes. VOP 28A has target sites at −573 in the VEGF-A gene and +61 inthe VEGF-C gene; while VOP 30A has a target sites at +42 and +530 inVEGF-A and one at −481 in VEGF-C.

HEK 293 cells were transfected with a plasmid encoding either a VOP28A-VP16 fusion or a VOP 30A-VP16 fusion, according to the methodsdescribed in Example 1, supra. Forty hours after co-transfection, VEGF-AmRNA was quantitated by TagMan® as described in Example 1, using theprimers and probes shown in Table 5. VEGF-C mRNA was quantitated in thesame RNA samples, according to the methods disclosed in Example 1, usingthe primers and probe disclosed in Table 8. The results, shown in FIG.14, indicate that each of the VOP 28A and VOP 30A fusion proteinsactivate production of both VEGF-A and VEGF-C mRNA.

As a control, HEK 293 cells were transfected with a plasmid encoding aVOP 32B-VP16 fusion. VOP 32B has a target site in the VEGF-A gene, at+434, but has no target site in the VEGF-C gene. Strong activation ofVEGF-A transcription, but no change in VEGF-C mRNA levels, was observedin these cells (FIG. 14).

These results show that multiple VEGF genes can be regulated by a singleZFP, leading to more efficient regulation of angiogenesis.

Example 6 In Vivo Induction of VEGF in Mice Using VEGF-Targeted ZFPFusions and Stimulation of Angiogenesis and Wound Healing I.Introduction

This series of experiments was designed to demonstrate further theability of appropriately designed ZFP fusion proteins to activateexpression of VEGF in vivo, and to show the ability of such proteins tostimulate angiogenesis and wound healing in mouse model systems that areused as models of the corresponding processes in humans.

Engineered ZFPs and knowledge of chromosomal structure were utilized ina variety of different types of investigations to achieve selectiveactivation of VEGF A. As in Example 1, certain VEGF stimulating ZFPswere designed following identification of targets by. DNAse Ihypersensitive mapping analysis. These initial experiments led to theidentification of three different DNAse-I accessible regions in bothNIH3T3 cells and C127I cells. ZFPs were designed to bind to either a9-bp or 18-bp target site within the accessible regions identified ineach cell type. Each of these designed ZFPs was shown to bind tightly toits intended target with an apparent K_(d) of <0.6 nM and more tightlythan a naturally occurring transcription factor (SP1).

Fusion proteins containing one of the designed ZFPs and the VP16transcriptional activation domain (Sadowski et al. (1988) Nature 335:563-564) were then tested for their ability to activate expression ofthe endogenous VEGF-A gene in C 127I cells at both the transcript andprotein levels. As described more fully infra, the results demonstratethat the designed ZFPs were able to activate the endogenous VEGF-A locusand, in so doing, accelerate processes of angiogenesis,reepithelialization and wound healing.

II. Experimental Procedures

A. Design of VEGF Regulating Zinc Finger Proteins

Mapping of DNAse I—Accessible Chromatin Regions in the Mouse VEGF-ALocus. C127I and NIH3T3 cells were obtained from the American TypeCulture Collection and maintained essentially as recommended by thesupplier. Nuclei were isolated and treated with DNAse I (Worthington)essentially as described (Zhang et al. (2000) J. Biol. Chem. 275:33,850-33,860; and Liu, et al. (2001) J. Biol. Chem. 276:11,323-11,334), except that DNAse I digests were for 1.5 min at 22° C.and the concentrations of DNAse I used were as indicated in the legendto FIG. 15A. Genomic DNA isolation, restriction enzyme digestion andSouthern blot analysis were then performed essentially as described(Zhang et al., supra; and Liu, et al., 2001, supra), except that enzymesand probes were as indicated in FIG. 15A.

Synthesis of genes encoding zinc finger proteins. The assembly of genesencoding the three-finger ZFPs mVZ+426 and mVZ+509 has been described(Liu et al., 2001, supra). [These designs were referred to,respectively, as VZ+434 and VZ+42/+530 in that study.] Briefly, oligosencoding α-helix and β-sheet regions of each three-finger protein wereassembled using PCR and each resultant ZFP gene was cloned into thepMal-c2 plasmid (New England Biolabs) as a fusion with DNA encodingmaltose binding protein.

To assemble the gene encoding the six-finger protein mVZ+57, thefollowing two-step strategy was utilized. First, genes encoding threefinger proteins corresponding to fingers 1-3 and 4-6 of VZ+57 wereconstructed and cloned as above, yielding constructs pMal-c2 ‘1-3’ andpMal-c2 ‘4-6’. Next, these two genes were joined via a short DNA spacerencoding a flexible peptide linker. This was accomplished as follows:(i) PCR of the ‘4-6’ ZFP gene using the primers 5′CCCAGATCTGGTGATGGCAAGAAGAAGCAGCACCATCTGCCACATCCAG (SEQ ID NO:241) and 5′CCCAAGCTTAGGATCCACCCTTCTTGTTCTGGTGGGT (SEQ ID NO:242); (ii) digestion ofthe resultant fragment with Bgl II and Hind III (sites underlined inprimers); and (iii) ligation into the BamHI and Hind III sites of thepMal-c2 ‘1-3’. The resultant protein, VZ+57, consists of the ‘1-3’ and‘4-6’ three-finger modules connected by a flexible peptide linker, withthe amino acid sequence between the second zinc-coordinating histidineof finger 3 and the first zinc-coordinating cysteine of finger 4 (bothunderlined) as follows: HQNKKGGSGDGKKKQHIC (SEQ ID NO:243).

Binding studies. Binding studies were performed essentially as described(Liu, et al. (2001) J. Biol. Chem. 276: 11,323-11,334), except that thebuffer was modified to the following final composition: 10 mM Tris, 100mM KCl, 1 mM MgCl₂, 10 mM DTT, 0.01 mM ZnCl₂, 10% glycerol, 200 μg/mlbovine serum albumin, 0.02% IGEPAL. Under these conditions SP1 (anaturally occurring zinc finger containing transcription factor used asa control) exhibits a significantly higher affinity than was determinedin previous studies (Liu et al., 2001, supra) and it is likely that theuse of our refined binding buffer contributed to the difference inapparent K_(d). ZFP concentrations for these studies were determineddirectly by measuring the DNA-binding activity of each ZFP preparationusing conditions under which binding is essentially stoichiometric(i.e., concentrations of ZFP and target site >50×K_(d)).

Construction of retroviral vectors. The retroviral vectors describedhere are derived from a pLXSN, a Moloney murine leukemia virus-basedvector containing a neomycin resistance gene under the control of aninternal simian virus (SV40) promoter. Using EcoR1 and Xho1 restrictionsites, the zinc finger expression cassette was placed immediatelydownstream of the LTR in pLXSN. Briefly, all ZFP constructs contained anN-terminal nuclear localization signal (Pro-Lys-Lys-Lys-Arg-Lys-Val)(SEQ ID NO:224) from SV40 largeT antigen, a Zinc Finger DNA-bindingdomain, the herpes simplex virus VP16 activation domain from amino acid413 to 490, and a FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) (SEQ IDNO:225). The LXSN vectors were produced in the 293 AMPHO-PAK™ cell lineand had titers ranging from 0.5−1.0×10⁶ G418-resistant colony-formingunits. Virus-containing supernatant was collected 48 hr aftertransfection, filtered through 0.45-mm-pore-size filter and used freshfor transduction of target cells or aliquoted and stored at −80° C.

Retroviral transduction. The AMPHO-PAK™ 293 cell line was obtained fromClontech and grown according to the manufacturer's recommendation. C127Icells were obtained from the American Type Culture Collection (ATCC) andgrown in Dulbecco's modified Eagle's medium (DMEN) supplemented with 10%fetal bovine serum. The cells were plated in 6-well plates at a densityof 2×10⁴ cells per well and then exposed 24 hour later to 2.0 ml ofvirus-containing supernatant in the presence of 8 μg/ml Polybrene, andincubated in a 37° C. incubator. The virus was removed 24 hour later andfresh medium was added. The cells were split 2 days later and G418containing medium was added. A G418-resistant population of cells wasestablished 14 days later and tested for VEGF expression by ELISA. Thecells were plated at a density of 5×10⁵ cells per well, refed at 24hours and media was collected for ELISA after 24 hours.

B. Adenovirus Construction and Experiments

Construction. Recombinant E1A/E1B deleted adenovirus vectors encodingzinc finger protein (ZFP) cDNA's were produced using the Ad-Easy system(T.-C. He, et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 2509-2514).Briefly, cDNA's encoding VEGF regulating ZFP's were subcloned into thepAd-Track-CMV shuttle vector. This vector contains two separate CMVpromoters, one to direct expression of inserted cDNA's and the other todirect expression of the transduction marker green fluorescent protein(GFP). In this shuttle vector, both the green fluorescent protein andthe cDNA expression cassettes are flanked by genomic adenovirus-5sequences, allowing recombination with genomic adenovirus-5 DNA (T.-C.He, et al. (1998) supra). More specifically, ZFP-encoding inserts frompcDNA MVG (a plasmid encoding the zinc finger binding domain VG10A/8A asdescribed in Example 2 supra) and three-finger constructs pCV VOP 30Aand pCV VOP 32B were cut by restriction digestion with EcoRI/HindIII andsub-cloned into shuttle plasmid pAdTrack-CMV, linearized with PmeI.

Each construct was co-electroporated along with the adenovirus-5 genomicvector pAd-Easy-1 into electrocompetent BJ5183 E. coli bacteria.Adenovirus-5 genomic DNA clones encoding the VEGF regulating ZFP's anddeficient for the essential adenovirus E1A/E1B gene region were obtainedby recombination between the ZFP encoding pAd-Track-CMV shuttle vectorsand pAd-Easy-1 in E. coli. Kanamycin resistant colonies were screenedfor recombinants by restriction mapping, and clones with the appropriaterestriction pattern were transfected into human embryonic kidney 293cells (HEK 293's) using the Lipofectamine reagent. HEK 293's contain theE1A/E1B genes and provide these essential proteins in trans to allowproduction and propagation of the replication defective E1A/E1Bdeficient adenovirus recombinants. These recombinants do not propagatein normal mammalian cells due to the lack of E1A/E1B gene functions.Adenovirus stocks were expanded in 293 cells, purified by CsClultracentrifugation, desalted by column purification or dialysis againstphosphate buffered saline, and titered on HEK 293 monolayers asdescribed (see, e.g., Giordano, et al. (1996) Nat. Med. 2:534-9; andGiordano et al. (1997) Circulation 96:400-3). Adenovirus stocks werestored in 20% glycerol at −80° C.

In vitro testing. To document that the above adenovirus vectors werecapable of directing expression of the VEGF-A regulating ZFP's aftertransduction, studies were performed in cultured rat aortic smoothmuscle cells (SMC). As indicated above, the ZFP constructs used in thesestudies contain both a FLAG epitope and a VP-16 activation domain.Adenovirus-mediated expression of the ZFP constructs was thus evaluatedby Western blot analysis of proteins expressed in the SMC usingantibodies directed to the FLAG epitope and the VP-16 activation domain.Briefly, SMC were grown in culture to subconfluence and were transducedwith adenovirus vectors encoding either a ZFP or green fluorescentprotein (GFP) (control). Transduction was carried out at a multiplicityof infection of approximately 5. 48 hours after transduction the cellswere scraped and protein lysates obtained for Western blot analysisusing specific antibodies against either the FLAG epitope (FisherScientific) or VP-16.

C. Induction of the VEGF-A Gene in Skeletal and Cardiac Muscle byVEGF-ZFP's

Plasmid Injection Studies The ability of a number of different VEGF-Aregulating ZFPs to induce expression of the endogenous VEGF-A gene wastested in vivo in rat skeletal muscle. Initial studies were carried outusing plasmid DNA expression vectors encoding either a VEGF-A regulatingZFP fusion protein or a control protein containing the same peptidesequences absent the DNA binding domain. A commercially availableplasmid in which gene expression is directed by the CMV promoter(pcDNA-3; Clontech) was used as the backbone for construction of the ZFPencoding plasmids.

Fifty μg of purified plasmid DNA encoding a VEGF-A regulating ZFP wasdiluted into 50 μl of phosphate buffered saline (PBS) and aspirated intoa 1 cc syringe. A small incision was made in the skin overlying theadductor muscle of the hindlimb of a Sprague-Dawley rat, and the plasmidDNA was injected directly into the muscle through a 30 gauge needle. Thesite was marked by injecting a small amount of India ink at the point ofplasmid DNA delivery. Fifty μg of the control plasmid was injected intothe contralateral hindlimb adductor muscle using the same methods. Atday 3 or 6 after plasmid delivery, the rats were sacrificed and skeletalmuscle from the plasmid injection sites was harvested using a 1 cmdiameter punch bioptome. The muscle tissue was rinsed in ice-cold PBSand rapidly frozen in liquid nitrogen and used for subsequent VEGF-Aprotein expression analysis by Western blotting.

ZFP-directed VEGF-A protein expression was also evaluated in heartmuscle using a similar approach. Briefly, in anesthetized and ventilatedCD-1 mice, the heart was exposed by left lateral thoracotomy. Fifty μgof plasmid DNA encoding either a VEGF-ZFP or the control peptide wasinjected in a 50 μl volume into the apex of the heart. Injection wasdocumented by blanching of the injection site. The chest was closed andthe animal allowed to recover. Three days after injection the animalswere sacrificed and the heart removed. The apex was clipped, rinsed inice cold PBS, rapidly frozen in liquid nitrogen and used for subsequentVEGF-A protein expression analysis by Western blotting (data for theseexperiments is not shown). Similar techniques can be used with rats.

Adenovirus Injection Studies. Using methodology similar to that used forthe plasmid DNA injection studies, VEGF-ZFP encoding or control (greenfluorescent protein encoding) adenovirus vectors were injected into thehindlimb adductor muscle of CD-1 mice. Approximately 5×10⁸ pfu ofVEGF-ZFP encoding adenovirus was injected in a 50 volume of PBS into theadductor muscle using a 1 cc syringe and a 30 gauge needle, as above.The contralateral adductor muscle was injected with a green fluorescentprotein encoding control adenovirus. Three or six days after adenovirusinjection the skeletal muscle injection site was harvested as above forWestern blot analysis of VEGF-A protein expression. For those VEGF-ZFP'sthat were designed to bind to DNA sequences in both the murine and ratVEGF-A regulatory domains (VOP 30A, VOP 32B, BVO12), in vivo testing wasalso carried out in the rat hindlimb adductor muscle. These studies werecarried out essentially as above, although the volume of the injectatewas increased to 100 Related experiments were also conducted withplasmids in the same volume.

D. Murine Ear Angiogenesis Assay

To test the ability of the designed VEGF-ZFP's to induce angiogenesis invivo, angiogenesis in the murine ear after injection of adenovirusvectors encoding either VEGF regulating ZFP's or the control peptidegreen fluorescent protein was evaluated (see, e.g., Pettersson, A., etal. (2000) Laboratory Investigation 80:99-115). Briefly, using a 1 ccsyringe and a 30 gauge needle ˜3×10⁸ pfu of VEGF-ZFP encoding adenoviruswas injected subcutaneously into the mouse ear in a volume of ˜25 μlPBS. The contralateral ear was similarly injected with an equal amountof control (green fluorescent protein) adenovirus. Three and six daysafter injection, the ears were visually inspected and digitalphotographs obtained using the same settings at the same distances. Theanimals were sacrificed, the ears harvested and fresh frozen in OTC forimmunohistochemical analysis of angiogenesis.

Vessel Counts. Angiogenesis/vascularization in the mouse ear wasevaluated on fresh frozen tissue sections using an anti-lectin antibody(Vector Laboratories, Burlingame, Calif.) that is specific forendothelial cells (see, e.g., Christie, K. N. and Thomson, C. (1989) J.Histochem. Cytochem. 37:1303-1304). Briefly, frozen sections were fixedwith acetone, rinsed with PBS, and incubated for 2 hours with theanti-lectin antibody. Following three sequential wash steps, thesections were incubated for 2 hours with a secondary antibody linked toalkaline phosphatase and the slides were developed using a commercialalkaline phosphatase staining kit (Vecta Inc.). Vessel counts weredetermined on the basis of microscopic analysis of anti-lectinimmunostaining by an observer blinded to the identity of the sections.Five separate 40× microscopic fields were evaluated per section, and thenumber of lectin stained vessels per field was averaged.

E. Wound Healing

To evaluate the ability of the VEGF-A regulating ZFP's to augmentangiogenesis in a functional in vivo biologic assay we used a wellestablished model of cutaneous wound healing (see, e.g., Swift, M. E.,et al., (1999) Lab Invest. 79:1479-87). Highly reproducible bilateraldorsal cutaneous wounds were created in CD-1 mice by excision of a 5 mmcircle of skin using a 5 mm punch bioptome. Healing of these woundsinvolves production of granulation tissue, reepithelialization byingrowth of keratinocytes and an angiogenic response. At the time ofwound creation ˜5×10⁸ pfu of VEGF-ZFP encoding adenovirus was deliveredto the wound site by topical application in a volume of 20 μl PBS. Thecontralateral wound site was treated with a control adenovirus encodinggreen fluorescent protein.

On day 5, the wound sites were excised whole, carefully bisected,formalin fixed and embedded in paraffin blocks. Multiple serial sectionswere obtained and used for histologic and immunohistologic analysis.Wound reepithelialization was analyzed on hematoxylin and eosin stainedsections evaluated under light microscopy with 4× and 40× objectives.Micrographic images were captured with a SPOT CCD camera and importedinto Adobe Photoshop for analysis. Two parameters of reepithelializationwere evaluated: a) the distance from the wound edge to the leading edgeof keratinocyte ingrowth, and b) the distance between the leading edgesof keratinocytes growing in from opposite sides of the wound. Allmeasurements were made quantitatively using computer calipers, and allsections were evaluated by an observer blinded to the treatment.

Wound vessel counts were determined by immunostaining of wound sectionsusing the endothelial cell specific anti-lectin antibody describedabove. Vessel counts from 5 separate microscopic fields were made persection (100× magnification) and the results per section averaged.

F. Western Blot Analysis

For Western Blot analysis of ZFP induced VEGF expression, animals werekilled by euthanasia at the 3rd day after adenoviral-ZFP or controladenoviral-green fluorescent protein (GFP) injections. Skeletal musclesaround the injection sites were carefully removed and homogenized at 4°C. in lysis buffer containing Tris-HCl 50 mM (pH 8.0), NaCl 150 SDS0.1%, NP-40 1%, Na Desoxycholate (0.5%) and proteinase inhibitors. Aftera ten-minute centrifugation (10,000 g at 4° C.), the protein-containingsupernatant fraction was collected, a small part of each sample was usedto determine protein concentration (BioRad) and samples were diluted(1:1) with 2× loading buffer containing 50 mM DTT and denatured byboiling 5 min. Samples containing equal amounts of total protein wereloaded on and separated by electrophoresis through 12% SDS-PAGE gels(Tris-Glycine, Novex). Proteins in the gel were transferred tonitrocellulose membranes and the membranes were exposed to mousemonoclonal anti-VEGF antibody (RDI) to check the level of VEGFexpression, rabbit polyclonal anti-VP16 antibody (Clonetech) to checkexpression of ZFP-VP16 fusion protein, and mouse monoclonal anti-musclespecific actin antibody (NCL) as an internal standard. After severalwashes in TBST, the blots were incubated with horseradish peroxidase(HRP)-conjugated secondary antibody for 1 h at room temperature,followed by TBST washes, and developed with an enhanced chemiluminescentsubstrate for detection of HRP (Pierce).

III. Results

A. Mapping of DNase I Accessible Regions in the Mouse VEGF-A Locus

The strategy for designing ZFP transcriptional regulators involved thepreferential targeting of accessible regions within the locus ofinterest. Such regions, which are readily identified via mapping ofDNase I hypersensitive sites (Gross et al. (1988) Ann. Rev. Biochem. 57:159-197), are generally more accessible to macromolecules thansurrounding stretches of DNA, and these regions often comprise bindingsites for natural transcriptional regulators of the associated genes.Preferential targeting of such regions with designed ZFPs tends to yieldboth more effective regulation and greater potency of response (Liu etal. (2001) J. Biol. Chem. 276:11,323-11,334). Accordingly, we havemapped DNase I accessible regions in mouse VEGF-A locus. In both NIH 3T3cells and the C127 I cell line, we observe three regions of enhancedDNase I accessibility centered approximately on bases −550, +1, and +400(numbers relative to the start site of transcription) (FIGS. 15A and15B). The −550 and +1 regions each appear to span approximately 200 basepairs, while the +400 region is somewhat more diffuse and encompassesapproximately 300 base pairs (FIGS. 15A and 15B). We have also observedsimilar patterns of DNase 1 accessibility in mouse TM3 cell line (datanot shown) and in a variety of cell types from man and rat (Liu et. al.,2001, supra).

B. Design and Biochemical Characterization of ZFPs Targeted to OpenChromatin Regions of Mouse VEGF-A

We next chose target sites within the ‘+1’ and ‘+400’ accessible regionsand designed zinc finger proteins that recognized these sequences withhigh affinity (FIG. 15C). This was accomplished by linking togetherfingers of known triplet preference to yield either three- or six-fingerZFPs with the desired sequence specificities. Designs for our ZFPs areshown in FIG. 15C, with the ZFPs named according to their targetlocation relative to the transcription start site of mouse VEGF-A. Twoof these designs, mVZ+426 and mVZ+509, contain three fingers and targetnine base pair sequences conserved in man and mouse. Our third ZFP,mVZ+57, contains six fingers and targets an 18 base pair sequencepresent in mouse (but not man).

Genes for our ZFPs were assembled (see experimental procedures) and eachprotein was expressed and purified essentially as described (Liu et.al., 2001, supra; and Zhang et. al., supra). We then characterized theDNA-binding affinity of each ZFP using a gel shift assay. Procedures forthese studies were essentially identical to those described previously(Liu et. al., 2001, supra; and Zhang et. al., supra) except for the useof a modified binding buffer. We found that our designed ZFPs bound totheir targets with high affinity, with K_(d)'s of 0.031 nM (FIG. 15D).For comparison, SP1, the parent ZFP for our designs, exhibited anapparent K_(d) of 0.053 nM for its DNA target under these conditions.

C. Activation of the Mouse VEGF-A Locus by ZFPs

We chose to use C127 I cells for our initial studies of VEGF-Aactivation, since this cell line exhibited a relatively low backgroundof endogenous VEGF-A expression against which to measure activationmediated by our designed ZFPs. For these studies, each ZFP was clonedinto pLXSN, a Moloney murine leukemia virus-based vector, as a fusionwith a VP16 activation domain, nuclear localization sequence, and FLAGtag. C127 I cells were then exposed to this vector, and transduced cellpopulations were selected using G418. As shown in FIG. 16B, expressionof our ZFP-VP16 fusions resulted in activation of the VEGF-A locus asdetermined by analysis of VEGF-A mRNA, with relative levels of VEGF-Amessage increasing by up to 4.6-fold for VZ+426-VP16 relative to acontrol construct expressing a VP16 activation domain fused to greenfluorescent protein. We also observed similar increases in the levels ofVEGF-A protein secreted into the medium as measured by ELISA (FIG. 16C).

D. Induction of the VEGF-A gene in Skeletal and Cardiac Muscle byVEGF-Binding ZFPs

In vitro characterization of adenovirus-mediated expression of VEGF-Aregulating ZFP's. Prior to in vivo testing of the recombinant adenovirusconstructs encoding the VEGF-A regulatory ZFP's, expression of the ZFP'swas documented by Western blot analysis. Aortic smooth muscle cells(SMC) were transduced with an adenovirus encoding either a VEGF-Aregulating ZFP or a green fluorescent protein (GFP). 48 hours aftertransduction the cells were washed with phosphate buffered saline andprotein lysates prepared. Western blot analysis using either ananti-VP16 antibody or an anti-FLAG epitope antibody revealed expressionof the ZFP constructs in adeno-VEGF-ZFP transduced cells, but not inadeno-green fluorescent protein (GFP) transduced cells (FIG. 17A).

VEGF-A protein expression after injection of recombinant adenovirusencoding VEGF-A regulating zinc finger protein VOP30A. Approximately5×10⁸ pfu of an adenovirus encoding a fusion protein comprising the VOP30A ZFP binding domain fused to a VP16 activation domain (Adeno-VOP 30A)was injected in a volume of 50 μl phosphate buffered saline into thehindlimb adductor muscle of CD-1 mice. The contralateral hindlimbadductor muscle was injected with adenovirus encoding green fluorescentprotein (adeno-GFP) as a control. Three days after the injection, themuscle encompassing the injection sites was harvested and used toprepare protein lysates for Western blot analysis of VEGF proteinexpression. As illustrated in FIG. 17B, adenovirus-mediated VOP30A geneexpression resulted in a marked induction of the VEGF-A gene in vivo asdemonstrated by the marked increase in VEGF-A protein expression.Western blotting with anti-VP16 antibody documents expression of theVOP30A construct in the injected muscle.

Induction of VEGF-A protein expression in mice after injection ofAdeno-MVG. Skeletal muscle injection of adeno-MVG or adeno-GFP (greenfluorescent protein) as a control was accomplished as just describedsupra. Three days after injection, VEGF-A protein expression wasevaluated by Western blot. Induction of VEGF-A protein expression wasobserved as shown in FIG. 17C. Equal loading of skeletal muscle proteinlysates is documented by immunostaining for actin.

VEGF-A protein expression in rats after skeletal muscle injection ofplasmids encoding VEGF-A regulating zinc finger proteins. Tests wereconducted in which plasmids encoding the ZFPs VOP 30A, VOP 32B, BVO12A,BVO14A or a control plasmid (lacking the ZFP binding domain) wereinjected into the hindlimb adductor muscle of Sprague-Dawley rats. Inall plasmids, protein expression was directed by the CMV promoter. Threedays after gene injection, the injection sites were excised. The resultsfrom Western blot analysis of the resulting protein lysates as shown inFIG. 17D show induction of VEGF-A protein by all four constructs.

E. Induction of Angiogenesis

Subcutaneous injection of recombinant adenovirus encoding VEGFregulating zinc finger proteins induces angiogenesis in the mouse ear.To assess the ability of VEGF-A regulating zinc finger proteins toinduce angiogenesis in vivo we injected recombinant adenovirus encodingVEGF-A regulating ZFP's subcutaneously into the mouse ear. Approximately5×10⁸ pfu were injected in a volume of 25 μl subcutaneously into the earof CD-1 mice. The contralateral ear was injected in the same manner witha control adenovirus encoding green fluorescent protein (GFP). Threedays and six days later the animals were visualized and digitalphotographs of the ears were taken. As shown in FIGS. 18A-D,adenovirus-mediated delivery of genes encoding VEGF-A regulating ZFP's(VOP30A or VOP 32B) resulted in augmented ear vascularization. Theseresults were correlated with data from formal vessel counts, as shown inFIG. 18E.

Increased vascularity is documented by vessel counts afteradenovirus-mediated delivery of genes encoding VEGF-A regulating zincfinger proteins. To further characterize the effects ofadenovirus-mediated delivery of genes encoding VEGF-A regulating ZFP'sin the ear angiogenesis assay just described, immunohistochemistryfacilitated vessel counts were performed on sections from control andVEGF-ZFP (VOP30A or VOP 32B) treated mouse ears. Vessel counts wereperformed by an observer blinded to the treatment group, and weredetermined by counting 5 separate fields per section at a magnificationof 40× and averaging the values (n=5 per group at day 3; n=4 per groupat day 6). As shown in FIG. 18E, assays conducted utilizing thisapproach also found a significant increase in vascularity for earsinjected with adenovirus encoding a ZFP (black box) as compared toadenovirus encoding green fluorescent protein (gray box).

Neovasculature resulting from activation of the endogenouse VEGF-A geneby the ZFP constructs is not hyperpermeable. Additional ear angiogenesisstudies used a modification of a previously described approach(Thurston, G., et al., 2000, Nature Medicine 6:460-463). Briefly,adenovirus vectors encoding either a VEGF-A activating ZFP or murineVEGF164 were injected subcutaneously in the ears of CD-1 mice (10⁸ pfuin 15 μl volume). Contralateral ears were injected with adenovirusencoding GFP. Digital photographs were obtained after three or six days.Evans Blue dye (200 μl of 4% solution) was injected by tail vein and thedistribution in the ears was assessed and photographed three hourslater. The mice were then sacrificed, ears embedded in OTC and frozen inliquid nitrogen cooled isopentane. 5 μm sections were fixed with coldacetone:methanol, immunostained with a monoclonal anti-PECAM antibodyand vessel counts obtained as previously described (Giordano, F. J. etal., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:5780-5785)

VEGF is a potent vascular permeability factor and has been shown toinduce hemorrhage and extravasation of intravascular dye in the mouseear model (Pettersson, A. et al., 2000, Lab. Invest. 80:99-115).Interestingly, when compared to ears similarly treated with anadenovirus encoding murine VEGF164, the ZFP-induced neovasculature wasnot spontaneously hemorrhagic and was not permeable to Evans Blue dyeinfusion (see FIG. 22). In other experiments, expression of angiopoietin1, a growth factor previously shown capable of promoting the growth of amore ‘mature’ non-leaky neovasculature, was not induced in the VEGF-ZFPtreated cells.

As shown in FIG. 22, the neovasculature resulting from ZFP-inducedexpression of VEGF-A expression was not hyperpermeable as was thatproduced by murine VEGF164 cDNA expression.

F. Accelerated Wound Healing

Cutaneous Wound Healing is Accelerated by VEGF-A Regulating ZFP's.

Bilateral cutaneous wounds were created in the backs of CD-1 mice byexcision of a 5 mm circle of skin using a punch bioptome. At the time ofwounding, adenovirus encoding a VEGF-A regulating zinc finger protein(MVG) was applied topically to the wound. The contralateral wound wastreated by topical application of a control adenovirus encoding greenfluorescent protein.

Shown in FIGS. 19A and 19B is an example of how treatment with theVEGF-A regulating ZFP augments the degree of reepithelialization notedat day 5 post-wounding. The arrows denote the leading edge ofkeratinocyte ingrowth into the wound. As is apparent, and as is shown inthe graph of FIG. 19C, the distance between the edges of keratinocyteingrowth is decreased by VEGF-ZFP treatment; thus reepithelialization isaugmented. All measurements were performed using captured digital imagesand a computer caliper program. The values shown in the graph arerelative units.

Wound reepithelialization is augmented by treatment with VEGF-Aregulating ZFP's. As depicted in FIGS. 20A and 20B, ingrowth of theleading edge of keratinocytes into the wound is augmented by the topicalapplication of recombinant adenovirus encoding a VEGF-A regulating ZFP(MVG). The arrowheads in the lower left of FIGS. 20A and 20B mark thewound edge and the upper arrow in each figure marks the extent ofkeratinocyte ingrowth at day 5 post-wounding. This analysis iscomplementary to the quantitation of the distance between thekeratinocyte ingrowth cones noted in FIGS. 19A and 19B.

Treatment of cutaneous wounds by topical application of recombinantadenovirus encoding VEGF-A regulating zinc finger proteins results inangiogenesis and increased vascularity. Cutaneous wounds were treated asjust described with either adenovirus encoding a VEGF-A regulating ZFP(MVG) or a control adenovirus encoding green fluorescent protein (GFP).Wounds were fixed, sectioned and immunostained with an antibody againstan endothelial cell specific lectin. As shown in FIGS. 21A and 21B,there is increased vascularity in the VEGF-ZFP treated wounds (FIG. 21A)as compared to the control wounds (FIG. 21B). Vessel counts wereperformed on digitally captured images and represent the average of 5high power fields per section (n=6 per group). The results with thisapproach (see FIG. 21C) are consistent with the immunostaining results.

IV. Discussion

The results of the experiments described in this example illustrate theability to design ZFPs capable of tightly binding to target sites withinthe promoter region of the VEGF-A gene utilizing the approachesdescribed herein. Certain of the ZFPs were found to bind more tightly totheir intended target site then the naturally occurring transcriptionfactor SP1. The ability of these ZFPs to function in vitro was shown bytransfecting C 127I cells with constructs encoding the ZFPs anddemonstrating increased expression of VEGF-A at both the transcript andexpressed protein level.

Using several different accepted model systems for angiogenesis andwound healing, the results from these investigations also show that ZFPscan be utilized to modulate angiogenesis and thus affect a wide varietyof conditions that are correlated with blood flow and blood delivery.More specifically, the foregoing results demonstrate that one canregulate angiogenesis by introducing plasmid or viral constructsencoding a ZFP having appropriate binding capability to modulateexpression of one or more VEGF genes in vivo and to thereby modulatevessel formation. As an example of the utility of this general approach,the results of certain of the model studies conducted in this exampleshow that introduced ZFPs can significantly accelerate woundreepithelialization and wound healing. Such utility was demonstratedusing accepted model systems and by established histological andimmunohistological methods. In view of these results, it is expectedthat the approaches illustrated in this example can be utilized in othertreatment applications that are based upon regulation of angiogenesis.By judicious selection of either an appropriate activation domain orrepressor domain with the ZFP, one can selectively increase or reduceangiogenesis depending upon the nature of the condition being treated.While this particular set of investigations was conducted using viral orplasmid constructs to introduce ZFPs into a cell, other delivery methodssuch as described supra can also be utilized. It is understood that theexamples and embodiments described herein are for illustrative purposesonly and that various modifications or changes in light thereof will besuggested to persons skilled in the art and are to be included withinthe spirit and purview of this application and scope of the appendedclaims. All publications, patents and patent applications cited hereinare hereby incorporated by reference in their entireties for allpurposes to the same extent as if each individual publication, patent orpatent application were specifically and individually indicated to be soincorporated by reference.

TABLE 1 Regions of VEGF Genes Examined for Potential Target Sites¹ A B²C D P1GF² Viral VEGF-E −2.3 −1.0 −0.60 −1.0 −1.0 −.23 +1.1 +0.32 +0.51+1.0 +1.0 +.62 ¹Numbers indicate the number of kilobase pairs upstream(negative number) or downstream (positive number) from thetranscriptional start site which were examined. ²The P1GF sequence, andthe portion of the VEGF-B sequence between −1.0 and −0.4, are based onhigh-throughput genome sequence data that may be subject to change.

TABLE 2 Locations of Target Site in VEGF Sequences VEGF E Viral ZFP NAMEVEGF A VEGF B VEGF C VEGF D (PlDGF) VEGF BVO 13A +851 EP10A −1083 −31−252 +534 GATA82Z7678 −485 −170 +183 HBV 3 +779 −245 HP38 4A −2248 −119+479 +805 −29 −1413 +510 +210 −1055 −633 HUM 17A −1002 −33 +472 HUM 19A−1016 MTS 5A −2251 +213 MX1E +851 PDF 5A +590 −748 RAT 24A +711 SAN 16A−1954 USX 3A +554 −230 +928 VEGF 1 −8 −454 −348 −36 VEGF 1*3 −8 −454−348 −36 VEGF 1A −8 −454 −348 −36 VEGF 1B −8 VEGF 1C −8 VEGF 1D −8 VG10A −1412 −774 −354 VG 1B −2252 −943 VG 4A −1083 −31 −252 VG 8A −2248−119 +479 +313 −903 +575 −633 −784 +510 +805 −29 −475 −22 −391 +179 +210VOP 28A-2 −573 +61 VOP 30A-4 +42 −481 +530 VOP 32A-6 +434 VOP 32B-7 +434VOP 35A-10 +892 ZEN-7A 1 −1273 −945 +61 −675 −573 BVO 10A-9A +621 BVO12A-11B +806 BVO 14B-13A +851 VOP 29A-3 +5 VOP 32C +434 VOP 32D +434 VOP32E +434 VOP 32F +434 VOP 32G +434 VOP 32H +434 VOP 32I +434 VOP 32J+434

TABLE 3 Target sites and recognition helix sequences of humanVEGF-targeted ZFPs ZFP SEQ. ID SEQ ID SEQ ID SEQ ID K_(d) NAME TARGET NOF 1 NO F 2 NO F 3 NO (nm) BVO 13A ATGGACGGG 1 RSDHLAR 30 DRSNLTR 59RSDALTQ 88 <.02 EP10A KGGGGCTGG 2 RSDHLTT 31 DRSHLAR 60 RSDHLSK 89 0.35GATA82Z678 GAGKGKGYG 3 RLDSLLR 32 DRDHLTR 61 RSDNLAR 90 1.8 HBV 3GGGGGAGGW 4 QTGHLRR 33 QSGHLQR 62 RSDHLSR 91 30 HP38 4A GGDTGGGGG 5RSDHLAR 34 RSDHLTT 63 QRAHLAR 92 0.75 HUM 17A ARGGGGGAG 6 RSDNLAR 35RSDHLSR 64 RSDNLTQ 93 <.02 HUM 19A TGGGCAGAC 7 DRSNLTR 36 QSGDLTR 65RSDHLTT 94 0.02 MTS 5A TGGGGGTGG 8 RSDHLTT 37 RSDHLTR 66 RSDHLTT 95 0.07MX1E ATGGACGGG 9 RSDHLAR 38 DRSNLTR 67 RSDALSA 96 3.4 PDF 5A GYAGGGGCC10 DRSSLTR 39 RSDHLSR 68 QSGSLTR 97 .23 RAT 24A GDGGAAGHC 11 ERGTLAR 40QSGNLAR 69 RSDALAR 98 <.02 SAN 16A AKGGAAGGG 12 RSDHLAR 41 QSGNLAR 70RSDALRQ 99 1.03 USX 3A GCCGGGGAG 13 RSDNLTR 42 RSDHLTR 71 DRSDLTR 1000.06 VEGF 1 GGGGAGGVK 14 TTSNLRR 43 RSSNLQR 72 RSDHLSR 101 2.83 VEGF 1*GGGGAGGVK 15 TTSNLRR 44 RSSNLQR 73 RSDHLSR 102 3 VEGF 1A GGGGAGGVK 16TTSNLRR 45 RSDNLQR 74 RSDHLSR 103 0.2 VEGF 1B GGGGAGGAT 17 QSSNLAR 46RSDNLQR 75 RSDHLSR 104 2 VEGF 1C GGGGVGGAT 12 TTSNLAR 47 RSDNLQR 76RSDHLSR 105 1 VEGF 1D GGGGAGGMT 19 QSSNLRR 48 RSDNLQR 77 RSDHLSR 106 2VG 10A GAWGGGGGC 20 DSGHLTR 49 RSDHLTR 78 QSGNLTR 107 ND VG 1B ATGGGGGTG21 RSDALTR 50 RSDHLTR 79 RSDALTQ 108 ND VG 4A GGGGGCTGG 22 RSDHLTT 51DRSHLAR 80 RSDHLSR 109 ND VG 8A GDGTGGGGN 23 QSSHLAR 52 RSDHLTT 81RSDALAR 110 .35 VOP 28A-2 GGGGGCGCT 24 QSSDLRR 53 DRSHLAR 82 RSDHLSR 111<.02 VOP 30A-4 GCTGGGGGC 25 DRSHLTR 54 RSDHLTR 83 QSSDLTR 112 <.02 VOP32A-6 GGGGGTGAC 26 DRSNLTR 55 MSHHLSR 84 RSDHLSR 113 <.02 VOP 32B-7GGGGGTGAC 27 DRSNLTR 56 TSGHLVR 85 RSDHLSR 114 <.02 VOP 35A-10 GCTGGAGCA28 QSGSLTR 57 QSGHLQR 86 QSSDLTR 115 <.02 ZEN-7A 1 GGGGGHGCT 29 QSSDLRR58 QSSHLAR 87 RSDHLSR 116 .63 VOP29A-3 GAGGCTTGG 244 RSDHLTT 51 QSSDLTR112 RSDNLTR 42 <.02 VOP 32-C GGGGGTGAC 26 DRSNLTR 55 TSGHLTR 245 RSDHLSR68 ND VOP 32-D GGGGGTGAC 26 DRSNLTR 55 TSGHLIR 246 RSDHLSR 68 ND VOP32-E GGGGGTGAC 26 DRSNLTR 55 TSGHLSR 247 RSDHLSR 68 ND VOP 32-FGGGGGTGAC 26 DRSNLTR 55 TSGHLAR 248 RSDHLSR 68 ND VOP 32-G GGGGGTGAC 26DRSNLTR 55 TSGHLRR 249 RSDHLSR 68 ND VOP 32-H GGGGGTGAC 26 DRSNLTR 55TAGHLVR 250 RSDHLSR 68 ND VOP 32-I GGGGGTGAC 26 DRSNLTR 55 TTGHLVR 251RSDHLSR 68 ND VOP 32-J GGGGGTGAC 26 DRSNLTR 55 TKDHLVR 252 RSDHLSR 68 ND

TABLE 4 Target sites and recognition helix sequences of humanVEGF-targeted ZFPs SEQ SEQ SEQ SEQ SEQ SEQ SEQ ZFP ID ID ID ID ID ID IDNAME TARGET NO F 1 NO F 2 NO F 3 NO F 4 NO F 5 NO F 6 NO BVOGTGGAGGGGGTCGGGGCT 117 QSSDLRR 120 RSDHLTR 123 DRSALAAR 126 RSDHLAR 129RSDNLAR 132 RSDALTR 135 10A- 9A BVO GGAGAGGGGGCYGCAGTG 118 RSDALTR 121QSGDLTR 124 ERGDLTR 127 RSDHLAR 130 RSDNLAR 133 QSGHLQR 136 12A- 11B BVOATGGACGGGtGAGGYGGYG 119 RSDELTR 122 RSDELTR 125 RSDNLAR 128 RSDHLAR 131DRSNLTR 134 RSDALTQ 137 14B- 13A

TABLE 6 Vegf-targeted ZFPs Target ZFP Sequence SEQ. ID Subsites FingerSEQ ID Apparent Name 5′-3′ NO: 5′-3′ Designs NO: Kd (nM) VZ −950GAAGAGGACc 138 GACc EKANLTR 149 0.18 GAGg RSDNLTR 150 GAAg QRSNLVR 151VZ −573 GGGGGCGCTc 139 GCTc QSSDLRR 152 0.63 GGCg QSSHLAR 153 GGGgRSDHLSR 154 VZ −475 GTGTGGGGTt 140 GGTt QSSHLAR 155 0.35 TGGg RSDRLTT156 GTGt RSDALAR 157 SP1 GGGGCGGGGg 141 GGGg KTSHLRA 158 0.25 (−76/+527)GCGg RSDELQR 159 GGGg RSDHLSK 160 VZ −8 GGGGAGGATc 142 GATc TTSNLRR 1612.83 GAGg RSSNLQR 162 GGGg RSDHLSR 163 VZ GCTGGGGGCt/g 143 GGCt/gDRSHLTR 164 <0.02 +42/+530 GGGg RSDHLTR 165 GCTg QSSDLTR 166 VZ +434bGGGGGTGACc 144 GACc DRSNLTR 167 <0.02 GGTg TSGHLVR 168 GGGg RSDHLSR 169VZ +434a GGGGGTGACc 145 GACc DRSNLTR 170 <0.02 GGTg MSHHLSR 171 GGGgRSDHLSR 172 VZ +472 AAGGGGGAGg 146 GAGg RSDNLAR 173 0.006 GGGg RSDHLSR174 AAGg RSDNLTQ 175 VZ +590 GCAGGGGCCg 147 GCCg DRSSLTR 176 0.23 GGGgRSDHLSR 177 GCAg QSGSLTR 178 VZ +892 GCTGGAGCAc 148 GCAC QSGSLTR 1790.24 GGAg QSGHLQR 180 GCTg QSSDLTR 181

TABLE 7 Target sites and recognition helix sequences of ratVEGF-targeted ZFPs ZFP NAME TARGET LOCATION RECOGNITION HELICES BVO 12A-11A GGAGAGGGGGCCGCAGTG +785 F1: RSDALTR (SEQ ID NO: 186) (SEQ ID NO:182) F2: QSGDLTR (SEQ ID NO: 187) F3: ERGDLTR (SEQ ID NO: 188) F4:RSDHLAR (SEQ ID NO: 189) F5: RSDNLAR (SEQ ID NO: 190) F6: QSSHLAR (SEQID NO: 191) BVO 14A- 13B ATGGACGGGtGAGGCGGCG +830 F1: RSDELTR (SEQ IDNO: 192) (SEQ ID NO: 183) F2: RSDELQR (SEQ ID NO: 193) F3: RSDNLAR (SEQID NO: 194) F4: RSDHLAR (SEQ ID NO: 195) F5: DRSNLTR (SEQ ID NO: 196)F6: RSDALTQ (SEQ ID NO: 197) VOP 32A GGGGGTGAC +420 F1: DRSNLTR (SEQ IDNO: 198) (SEQ ID NO: 184) F2: MSHHLSR (SEQ ID NO: 199) F3: RSDHLSR (SEQID NO: 200) VOP 30A GCTGGGGGC  +40 F1: DRSHLTR (SEQ ID NO: 201) (SEQ IDNO: 185) +514 F2: RSDHLTR (SEQ ID NO: 202) F3: QSSDLTR (SEQ ID NO: 203)VOP 32B GGGGGTGAC +420 F1: DRSNLTR (SEQ ID NO: 198) (SEQ ID NO:184) F2:TSGHLVR (SEQ ID NO: 85) F3: RSDHLSR (SEQ ID NO: 200)

TABLE 8 Nucleotide sequences of probe and primers used for analysis ofVEGF-C mRNA SEQ ID SEQUENCE NO VEGF-C-Forward 5′-TGCCGATGCATGTCTAAACT-3′204 primer VEGF-C-Reverse 5′-TGAACAGGTCTCTTCATCCAGC-3′ 205 primerVEGF-C-Probe 5′-FAM-CAGCAACACTACCACAGTGTCAGG 206 CA-TAMRA-3′

1-8. (canceled)
 9. A method for modulating angiogenesis comprisingintroducing a nucleic acid encoding a zinc finger protein into an animalhaving a genome comprising a target site within a vascular endothelialgrowth factor (VEGF) gene, whereby the zinc finger protein binds to thetarget site and thereby modulates angiogenesis in the animal, whereinthe zinc finger protein binds to a target site specified in Table 3 (SEQID NOS: 1-29 and 244) or Table 4 (SEQ ID NOS: 117-119).
 10. The methodaccording to claim 9, wherein positions −1 to +6 in each of three zincfingers are occupied by first (SEQ ID NOS: 30-58), second (SEQ ID NOS:59-87, 112, and 245-252) and third segments (SEQ ID NOS: 42, 68 and88-116) of seven contiguous amino acids as specified in a row of Table3.
 11. The method according to claim 9, wherein the zinc finger proteincomprises six zinc fingers, and positions −1 to +6 in each of the sixzinc fingers are occupied by first (SEQ ID NOS: 120-122), second (SEQ IDNOS: 123-125), third (SEQ ID NOS: 126-128), fourth (SEQ ID NOS:129-131), fifth (SEQ ID NOS: 132-134) and sixth (SEQ ID NOS: 135-137)segments of seven contiguous amino acids as specified in a row of Table4.
 12. The method according to claim 9, wherein the target site ispresent in a plurality of VEGF genes, whereby the zinc finger proteinbinds to the target site in the plurality of genes, thereby modulatingexpression of the plurality of VEGF genes.
 13. A method of treatingischemia, comprising administering a nucleic acid encoding a zinc fingerprotein that binds to a target site within a VEGF gene into an animalhaving ischemia, wherein the animal has a genome comprising a VEGF genecomprising the target site and the zinc finger protein binds to thetarget site, and wherein the zinc finger protein is administered in anamount effective to treat ischemia, wherein the zinc finger proteinbinds to a target site specified in Table 3 (SEQ ID NOS: 1-29 and 244)or Table 4 (SEQ ID NOS: 117-119).
 14. The method according to claim 13,wherein positions −1 to +6 in each of three zinc fingers are occupied byfirst (SEQ ID NOS: 30-58), second (SEQ ID NOS: 59-87, 112, and 245-252)and third segments (SEQ ID NOS: 42, 68 and 88-116) of seven contiguousamino acids as specified in a row of Table
 3. 15. The method accordingto claim 13, wherein the zinc finger protein comprises six zinc fingers,and positions −1 to +6 in each of the six zinc fingers are occupied byfirst (SEQ ID NOS: 120-122), second (SEQ ID NOS: 123-125), third (SEQ IDNOS: 126-128), fourth (SEQ ID NOS: 129-131), fifth (SEQ ID NOS: 132-134)and sixth (SEQ ID NOS: 135-137) segments of seven contiguous amino acidsas specified in a row of Table
 4. 16. The method of claim 13, whereinthe zinc finger protein is applied to a specific tissue of the animal.17. The method according to claim 13, wherein the zinc finger proteincomprises at least three fingers of the C2H2 class of zinc fingers. 18.A method for treating a wound comprising introducing a nucleic acidencoding a zinc finger protein into an animal having a genome comprisinga target site within a VEGF gene, whereby the zinc finger protein bindsto the target site, such binding accelerating healing of the wound, andwherein the zinc finger protein binds to a target site specified inTable 3 (SEQ ID NOS: 1-29 and 244) or Table 4 (SEQ ID NOS: 117-119). 19.The method according to claim 18, wherein positions −1 to +6 in each ofthree zinc fingers are occupied by first (SEQ ID NOS: 30-58), second(SEQ ID NOS: 59-87, 112, and 245-252) and third segments (SEQ ID NOS:42, 68 and 88-116) of seven contiguous amino acids as specified in a rowof Table
 3. 20. The method according to claim 18, wherein the zincfinger protein comprises six zinc fingers, and positions −1 to +6 ineach of the six zinc fingers are occupied by first (SEQ ID NOS:120-122), second (SEQ ID NOS: 123-125), third (SEQ ID NOS: 126-128),fourth (SEQ ID NOS: 129-131), fifth (SEQ ID NOS: 132-134) and sixth (SEQID NOS: 135-137) segments of seven contiguous amino acids as specifiedin a row of Table 4.